Compositions and methods for protecting the kidney from ischemia reperfusion injury

ABSTRACT

The effects of hepcidin treatment on mitigating ischemia reperfusion injury (IRI) and acute kidney injury (AKI) by decreasing iron availability and ROS-mediated cell death were tested. Wild type (WT) C57Bl/6 and hepcidin knock out (Hamp −/− ) mice were treated with saline or 50 μg of hepcidin i.p. prior to bilateral renal IRI. Renal function, injury markers, histopathology, and inflammation were examined after 24 hours of reperfusion. In WT mice, IRI induced increases in serum and kidney non-theme iron levels, but hepcidin treatment induced sequestration of iron in the spleen and liver and prevented IRI-associated increases in serum and kidney non-heme iron. Kidney function was significantly better in hepcidin-treated mice, accompanied by less acute tubular necrosis and reduced infiltration of immune cells. Hepcidin treatment decreased kidney ferroportin expression and induced the expression of cytoprotectant, H-Ferritin, and was associated with less ROS and tubular epithelial apoptosis. These results demonstrate a protective role of hepcidin in IRI and AKI.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 15/026,574, filed Mar. 31, 2016, which is a national stage filing ofInternational Application No. PCT/US2014/056751, filed Sep. 22, 2014,which claims benefit of priority pursuant to 35 U.S.C. § 119(e) to U.S.provisional patent application No. 61/880,272, filed on Sep. 20, 2013.The entire disclosures of the afore-mentioned patent applications areincorporated herein by reference.

BACKGROUND

Renal ischemia-reperfusion injury (IRI) is a major cause of acute kidneyinjury (AKI) in many clinical settings including cardiovascular surgery,sepsis, and kidney transplantation. Ischemic AKI is associated withincreased morbidity, mortality, and prolonged hospitalization (1, 2).

Acute ischemia leads to depletion of adenosine triphosphate (ATP),inducing tubular epithelial cell (TEC) injury, and hypoxic cell death.Reperfusion further amplifies injury by promoting the formation ofreactive oxygen species (ROS), and inducing leukocyte activation,infiltration, and inflammation (3-6).

Multiple studies have shown the important role of iron and ROS inmediating apoptotic and necrotic cell death and the ensuing inflammatoryresponse during the course of IRI (7, 8). The ischemic stage of theinjury results in mitochondrial membrane depolarization, DNAfragmentation, translocation of cytochromes from the mitochondria intothe cytosol (9, 10) and breakdown of heme proteins like the cytochrome cresults in a reduction of heme associated ferric (Fe³⁺) iron to ferrous(F²⁺) iron and thereby increase the levels of catalytically active orlabile form of iron (11-13). A key pathological characteristic of labileiron (Fe²⁺) is its ability to catalyze the generation of tissue damaginghydroxyl radical (OH—) by an interaction with superoxide anion (O⁻²) andhydrogen peroxide (H₂O₂) via Haber-Weiss reaction, both of which areincreased during IRI (7, 14). Labile iron's contribution to oxidativestress and cellular damage has been demonstrated not only in renalischemia reperfusion injury (15, 16) but also in other models of AKI(17, 18) and iron chelation with desferrioxamine induces protection indiverse animals models of AKI (19).

Iron (Fe²⁺) is exported from the cells by the only known iron exportprotein, ferroportin (20). Ferroportin is significantly expressed onmacrophages, hepatocytes, renal proximal and distal tubular cells, andenterocytes (21-23). Intracellular iron levels regulate ferroportinexpression; high intracellular iron induces ferroportin-mediated ironexport into circulation (20). To sustain physiologic iron requirementyet avoid iron toxicity, an endogenous peptide hormone Hepcidin (HAMP),primarily produced by hepatocytes (23, 24), and regulates systemic ironbalance. The main known function of HAMP is to covalently modifyferroportin, which leads to its internalization and lysosomaldegradation, and thereby prevent cellular iron egress (25, 26). HAMP isacutely and positively regulated during iron imbalance (25),inflammation (27, 28) and has antibacterial properties (29, 30; see also60 and 61). Hypoxia, however, negatively regulates it (31). While humanstudies have indicated a positive correlation between increased urinaryHepcidin levels and protection against AKI, a direct pathogenic role ofhepcidin has not been examined in any model of AKI (32).

Mature hepcidin is a 25 amino acid (a.a.) residue peptide. Itsproduction appears to be regulated at the transcriptional level andmajor stimuli regulating hepcidin production include iron and theregulatory signals pertaining to erythropoietic demands for iron.Hepcidin is also an acute-phase reactant and increases duringinflammation. Other hepcidin regulators include hepatocyte growth factor(HGF), epidermal growth factor (EGF), steroid hormones (estrogen,testosterone), and metabolic pathways (starvation/gluconeogenesis).Hepcidin production increases in response to iron loading and thisprevents further absorption of dietary iron and the development of ironoverload. Plasma iron and liver iron stores regulate hepcidintranscription. Both serum iron and liver iron accumulation activate theBMP receptor and its Smad1/5/8 pathway, and increase hepcidin mRNAconcentrations in hepatocytes. The BMP co-receptor hemojuvelin (HJV) isalso required for this response.

For extracellular iron, transferrin receptors 1 and 2 (TfR1 and TfR2)are the likely sensors of holo-transferrin concentrations. At higherholo-Tf concentrations, HFE is displaced from TfR1 and associates withTfR2. HFE and TfR2 in turn may interact with HJV, thus potentiating BMPsignaling.

Mutations in Hfe, TfR2, Hjv, Bmp6, BMP receptors Alk2 and Alk3, andSmad4 all impair hepcidin regulation by iron. Hepcidin production isfurther modulated by the transmembrane serine protease TMPRSS6, alsoknown as matriptase-2, and by neogenin, a multifunctional transmembranereceptor. It has been proposed that these proteins act bypost-translationally regulating the levels of membrane-associated HJV.The specific involvement of these proteins in iron sensing is alsouncertain.

Hepcidin is suppressed in conditions associated with increasederythropoietic activity. Hemorrhage, hemolysis, and injections oferythropoietin all result in a rapid decrease in hepcidin. In anemiaswith ineffective erythropoiesis, hepcidin levels are chronicallysuppressed. This is thought to be the cause of iron overload innontransfused patients. Hepcidin increases rapidly followinginflammatory and infectious stimuli via the IL-6 pathway. Becausehepcidin deficiency or excess plays important roles in the pathogenesisof various iron disorders, hepcidin agonists and antagonists may bepotentially useful in clinical practice.

Hepcidin agonists such as PR73 (a minihepcidin) are compounds that canmimic the function of hepcidin or potentiate its endogenous synthesisand may be able to prevent systemic accumulation of iron (See Ganz etal., International Pat. Pub. WO2013086143 A1). Such compounds mayprovide additional treatment options for patients who do not respondwell to standard treatment regimens. Minihepcidins are peptide-basedhepcidin agonists that were rationally designed based on the region ofhepcidin that interacts with ferroportin. A nine amino acid N-terminalfragment of hepcidin (DTHFPICIF) is crucial for its hormonal activity.This particular fragment was further engineered: unnatural amino acids(N-substituted and β-homo amino acids) were introduced to increaseresistance to proteolysis, and fatty acids were conjugated to prolongthe half-life in circulation. This yielded analogs that are at least aspotent as full-length hepcidin and have a longer duration of action. Onesuch analog, the minihepcidin PR65, was tested in hepcidin knockoutmice, a model of severe hemochromatosis. Treatment prevented thedevelopment of iron overload in non-overloaded hepcidin knockout mice.Treatment of mice with pre-existing iron overload was less effective butstill led to partial redistribution of iron from the liver to the spleenwithin 2 weeks. At high doses, PR65 caused profound iron restriction andanemia, indicating that minihepcidin therapy will likely requiretitration to effect to avoid excessive hypoferremia and ironrestriction.

Hepcidin production can be increased by antagonizing TMPRSS6, a negativeregulator of hepcidin. Homozygous inactivation of Tmprss6 in thalassemicth3/+ mice increased hepcidin levels, ameliorated iron overload, andimproved ineffective erythropoiesis. Targeting Tmprss6 with RNA-basedtherapeutics such as antisense oligonucleotides (ASOs) and siRNAsagainst Tmprss6 was effective in a mouse model of iron overload.

Hepcidin production can also be stimulated by BMP6 and its agonists. Inpatients undergoing low-molecular-weight heparin therapy to prevent deepvein thrombosis, serum hepcidin concentrations decreased by ˜80% within2-5 days after the start of the treatment. This was associated withincreased serum iron and transferrin saturation. Heparin itself is ananti-inflammatory agent, which may be a contributory factor in itsanti-hepcidin activity. HJV, a BMP co-receptor essential for hepcidinexpression, is another molecular target that can be exploited tointerfere with hepcidin production. Membrane-linked HJV and its solubleform (shave) have opposing effects on hepcidin expression, and shavedecreases Smad signaling and hepcidin levels. Soluble HJV-Fc fusionprotein (sHJV.Fc) ameliorated anemia of inflammation (AI) in a rat modelin which AI was induced with group A streptococcalpeptidoglycan-polysaccharide (PG-APS). Four-week therapy resulted inincreased hemoglobin and serum iron, although hepcidin mRNA had notsignificantly decreased by this point.

LDN-193189, a derivative of dorsomorphin which specifically antagonizesthe kinase activity of BMP receptor isotypes ALK2, ALK3, and ALK6,effectively reversed anemia in the rat model of AI caused by PG-APS.

Inflammation induces hepcidin expression via IL-6-Stat3 and possiblyother pathways and neutralizing monoclonal antibodies directed againstIL-6 or IL-6 receptors can be used to decrease hepcidin synthesis inanimal models and humans with inflammatory conditions.

There is a long felt need in the art for compositions and methods usefulfor preventing and treating acute kidney injury associated with renalischemia reperfusion, including during surgery such as transplantsurgery. The present invention satisfies these needs.

SUMMARY OF THE INVENTION

It is disclosed herein that renal IRI induces unique dynamic changes inrenal and extra-renal iron homeostasis characterized byferroportin-dependent splenic iron egress and renal iron accumulation.Hepcidin knockout mice that demonstrate constitutive overexpression offerroportin sustain more severe renal injury after IRI. It is furtherdisclosed herein that synthetic Hepcidin mitigates renal IRI throughreducing circulating iron levels, by inducing hepatosplenic ironsequestration and by reducing inflammation and oxidative stress. Theresults disclosed herein suggest that hepcidin therapy, including theuse of synthetic fragments of hepcidin, may represent an effective novelstrategy for the mitigation of renal IRI. In one aspect, hepcidin isprotective against folic acid induced acute kidney injury (data notshown).

Acute kidney injury resulting from other causes can also be treated orprotected by hepcidin therapy and include, but are not limited to,myoglobinuric acute kidney injury, acute kidney injury induced byhypotensive shock, sepsis-associated acute kidney injury, toxic acutekidney injury such as after aminoglycoside therapy or cancerchemotherapy, acute kidney injury induced by iodinated contrast media(CI-AKI). CI-AKI is a common cause of acute kidney injury which can bepredicted by certain risk factors and hepcidin may have a usefulapplication.

Therefore, the present invention provides compositions and methodsuseful for preventing or treating acute kidney injury associated withrenal ischemia reperfusion injury and with other causes of acute kidneyinjury. The method comprises administering to a subject a pharmaceuticalcomposition comprising an effective amount of at least one hepcidin or abiologically active fragment or homolog thereof, and optionally apharmaceutically acceptable carrier. In one aspect, the compositionfurther comprises at least one inducer of hepcidin and optionally anadditional therapeutic agent.

The present invention provides for the use of hepcidin as describedherein, as well as biologically active fragments and homologs thereof(e.g., SEQ ID NOs:1-5). In one aspect, the hepcidin is mammalian. In oneaspect, the hepcidin is human. In one aspect, the hepcidin is synthetic.In one embodiment, hepcidin or biologically active fragments or homologsthereof are useful for protecting the kidney from injuries associatedwith renal IRI. The present invention provides for administering apharmaceutical composition to a subject in need, wherein the compositioncomprises hepcidin or a biologically active fragment or homolog thereof,and optionally a pharmaceutically-acceptable carrier and optionally atleast one additional therapeutic agent.

In another aspect, the administration of a hepcidin protein or peptideof the invention can be performed at the same time other therapies arebeing administered. One of ordinary skill in the art will appreciatethat numerous techniques are known for determining the best routes,dosages, and timing of administration, as well as how many timesadministration should occur.

The present invention provides compositions and methods useful forprotecting the kidney from injury associated with renal ischemiareperfusion injury. The treatments described herein are useful for,inter alia: preventing or treating oxidative stress associated injury inthe kidney; inhibiting increased serum iron levels associated withischemia reperfusion injury; inhibiting kidney iron accumulation;increasing splenic non-heme iron levels; inhibiting a decrease in livernon-heme iron; inhibiting increased hepcidin gene expression; inhibitingincreased endogenous serum hepcidin levels; inhibiting increased plasmacreatinine levels; inhibiting kidney tubular necrosis; inhibiting renalepithelial apoptosis; inhibiting oxidative stress; reducinginflammation; inhibiting infiltration of immune cells into the kidney;preventing or inhibiting an increase in ischemiareperfusion-injury-induced renal ferroportin levels; inhibiting adecrease in H-ferritin levels; inhibiting infiltration of neutrophilsand CD11b cells into the kidney; inhibiting splenic iron release;stimulating an increase in H-ferritin levels; and inhibiting an increasein interleukin-6 (IL-6) levels.

The present invention further provides inducers of hepcidin and theiruse as described herein. In one aspect, hepcidin inducers include, butare not limited to, iron, iron-hepcidin complex, copper-hepcidincomplex, metal-hepcidin complex, TMPRSS6 inhibition using antagonistsagainst Tmprss6, plant-derived flavonoids, cytokines (such as IL-6 andIL-22), growth factors such as HGF and EGF, steroid hormones such asestrogen and testosterone, toll-like receptor activation, tyrosinekinase inhibitors such as Sorafenib and mTOR inhibitors such asrapamycin, administration of BMP6, and induction of inflammation.Inducers of inflammation include, for example, erythroferrone (Fam132b)and heparin antagonists.

Other hepcidin inducers include, but are not limited to, agents thatinhibit growth factor receptor dependent signaling (AG1296, GTP 14564,AS252424, 10058-F, SU6668, and pterostilbene), decrease inflammation(leflunomide, amlexanox), or impair DNA repair and promote apoptosis(daunorubicin, 9-aminocridine, ethacridine), while the small molecules,vorinostat and SB 204741, inhibit histone deacetylase and serotoninreceptor 2B, respectively. Two of the molecules, ipriflavone andvorinostat, have been shown to be active at concentrations that are10-fold below those required for genistein's effect (Gaun et al., BloodCells Mol. Diseases, 2014; available online 4 Jul. 2014)

The invention further encompasses the use of synthetic hepcidinderivatives. The invention further encompasses the use of minihepcidinssuch as PR73 and PR65, as well as alpha-2 macroglobulin modifiedhepcidin which increases the circulating half-life of hepcidin. Theseinducers can be used as a combination therapy in conjunction withhepcidin. More than one inducer can be used. One of ordinary skill inthe art can determine whether all agents are administered at the sametime or if they should be administered at different times.

In one aspect, cytokines of the invention include IL-6 and IL-22. In oneaspect of the invention, growth factors include HGF and EGF. In oneaspect, steroids of the invention include estrogen and testosterone. Inone aspect, antagonists of TMPRSS6 include antagonists such as antisenseoligonucleotides and siRNAs against Tmprss6.

In one aspect, hepcidin treatment protects the kidney by degradingferroportin and causing sequestration of iron. In one aspect, iron issequestered predominantly in the spleen. In one aspect, the treatmentinduces reactive oxygen species, apoptosis, and immune cell infiltrationin the kidney.

The dosage of hepcidin, or a biologically active fragment or homologthereof (e.g., SEQ ID NOs:1-5), administered to a subject in needthereof can be determined by one of ordinary skill in the art. Forexample, in one aspect, it can be from about 0.1 mg/kg body wt. to about100 mg/kg body weight. In one aspect, it can be from about 1.0 mg/kgbody wt. to about 10 mg/kg body wt. Dosages and dosage ranges includeintervening integers not specifically recited. For example, 1 to 10includes 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, as well as fractions anddecimals thereof. Doses include, for example, about 1.0, 1.5, 1.67, 2.0,2.5, 3.0, 3.33, 4.0, 4.5, 5.0, and 10 mg/kg body wt. Unit doses are alsoencompassed by the invention, such as from 50 mg to 1,000 mg, forexample 50, 55, 60, 65, 70, 75, 100, 125, 150, 175, 200, 225, 250, 275,300, 325, 350, 400, 450, 500, or 1,000 mg. One of ordinary skill in theart can determine the dosage, number of doses, and timing of doses basedon the age, sex, weight, and health of the subject.

In one aspect, a pharmaceutical composition comprising an effectiveamount of a hepcidin of the invention, or fragments or homologs thereof,is administered at least twice. In another aspect, a pharmaceuticalcomposition is administered at least five times. In yet another aspect,a pharmaceutical composition is administered at least 10 times. One ofordinary skill in the art can determine how often to administer thecomposition based on the particular disease or disorder being treated orhow the subject has responded to prior treatments. One of ordinary skillin the art can also determine when to administer a treatment relative tothe time that an IRI event occurs, including before, after, or both.

In one embodiment, the subject is treated with hepcidin prior to the IRIevent. In one aspect, the subject can be treated starting at leastseveral days before the event or as close to several minutes before theIRI event. For example, the hepcidin therapy can begin at about 2 hours,8 hours, 24 hours, or 26 hours prior to IRI. One of ordinary skill inthe art will appreciate that hepcidin or biologically active fragmentsor homologs thereof can be administered at varying times and not just atabout 2, 8, 24, or 26 hours prior to IRI. The invention furtherencompasses treatment following the initiation of IRI. In one aspect,the range of time for treating prior to the IRI event can be from about1.0 minutes to about 72 hours. In another aspect, the range of time fortreating prior to the IRI event can be from about 10 minutes to about 48hours. In another aspect, the range of time for treating prior to theIRI event can be from about 30 minutes to about 24 hours.

In one embodiment, the subject is treated with hepcidin after the IRIevent or both before and after as described above. In one aspect, thesubject can be treated starting immediately after such as severalminutes after the IRI event. For example, the hepcidin therapy can beginat about 30 minutes, 2 hours, 8 hours, 24 hours, or 48 hours after theIRI. One of ordinary skill in the art will appreciate that hepcidin orbiologically active fragments or homologs thereof can be administered atvarying times as well.

In one embodiment, 2-glucosamine modified hepcidin can be administeredto increase renal selectivity.

The present invention further provides for the use of hepcidin inreducing proteinuria (data not shown). Therefore, the invention providesfor the use of hepcidin therapy in treating proteinuric kidney diseases,for example, diabetic nephropathy and glomerulonephritis. In one aspect,treatment reduces proteinuria. In one aspect, treatment prevents orinhibits progression of kidney disease.

Because the methods of the invention are useful for treating ischemicreperfusion injury, the methods further include treating other diseasesand disorders associated with ischemic reperfusion injury, including,but not limited to, myocardial IRI and brain IRI.

Summary of Sequences of the Invention—

The present invention provides for the use of various hepcidin proteinsand peptides with the activity described herein as well as biologicallyactive fragments and homologs thereof.

SEQ ID NO: 1 Mouse hepcidin 25 (amino acids 59 to 83 of SEQ IDNO: 4) from Peptide International has the sequence:DTNFPICIFCCKCCNNSQCGICCKT SEQ ID NO: 2a 25 a.a. fragment of human hepcidin SEQ ID NO: 3,and is the equivalent to the murine 25 a.a. peptide SEQ ID NO: 1.DTHFPICIFCCGCCHRSKCGMCCKT SEQ ID NO: 3Human hepcidin is an 84 amino acid residuepeptide. Its GenBank accession number is AAH20612.1.MALSSQIWAACLLLLLLLASLTSGSVFPQQTGQLAELQPQDRAGARASWMPMFQRRRRRDTHFPICIFCCGCCHRSKCGMCCKT SEQ ID NO: 4Mouse hepcidin is an 83 amino acid residuepeptide. Its GenBank accession number is NP_115930.1.MALSTRTQAACLLLLLLASLSSTTYLHQQMRQTTELQPLHGEESRADIAIPMQKRRKRDTNFPICIFCCKCCNNSQCGICCKT SEQ ID NO: 5Rat hepcidin precursor is an 84 amino acid residuepeptide. Its GenBank accession number is NP_445921.1MALSTRIQAACLLLLLLASLSSGAYLRQQTRQTTALQPWHGAESKTDDSALLMLKRRKRDTNFPICLFCCKCCKNSSCGLCCIT

In one aspect, the invention uses a biologically active hepcidin proteinor peptide. Preferably, the isolated polypeptide comprises a mammalianmolecule at least about 30% homologous to a polypeptide having the aminoacid sequence of at least one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3,SEQ ID NO:4, and SEQ ID NO:5. Preferably, the isolated polypeptide is atleast about 35% homologous, more preferably, about 40% homologous, morepreferably, about 45% homologous, even more preferably, about 50%homologous, more preferably, about 55% homologous, preferably, about 60%homologous, more preferably, about 65% homologous, even more preferably,more preferably, about 70% homologous, more preferably, about 75%homologous, even more preferably, about 80% homologous, more preferably,about 85% homologous, more preferably, about 90% homologous, even morepreferably, about 95% homologous, more preferably, about 96% homologous,more preferably, about 97% homologous, more preferably, about 98%homologous, and most preferably, about 99% homologous to at least one ofSEQ ID NOs:1-5.

The present invention further encompasses modification of the hepcidinproteins and peptides, including amino acid deletions, additions, andsubstitutions, as well as modifications to increase in vivo half-lifeand decrease degradation in vivo.

The present invention includes an isolated nucleic acid comprising anucleic acid sequence encoding a mammalian hepcidin protein, or afragment or homolog thereof. In one aspect, the sequence encodes apeptide comprising a sequence having SEQ ID NOs:1, 2, 3, 4, or 5, or abiologically active fragment of homolog thereof.

Synthetic hepcidins are encompassed by the invention and derivativesinclude minihepcidin.

The present invention further provides kits for treating subjects asdescribed herein, comprising a hepcidin protein or peptide, anapplicator, and an instructional material for the use thereof. Kits canbe provided with unit doses in a container or syringe or amounts ofhepcidins that one of ordinary skill in the art can administer based ona dose per weight, etc.

Various aspects and embodiments of the invention are described infurther detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1, comprising FIGS. 1a to 1f . Renal IRI is associated with changesin systemic iron levels and upregulation of endogenous Hepcidin, whichare prevented by exogenous Hepcidin. Serum iron was measured by ELISAand normalized to sample volume (a). Non-heme iron was measuredfollowing acid digestion of Kidney (b), Liver (c), and Spleen (d)normalized to tissue weight and expressed as micro gram per gram tissue.Liver RNA was isolated, cDNA was amplified and normalized to GAPDH (e).Hepcidin content in the serum was measured by ELISA (f). Sham (□), IRI(◯) and Hepcidin+IRI (∇). *P<0.05, **P<0.005, ***P<0.0005, ****P<0.0001.Data points are plotted as mean±SEM (n=4-5 per group).

FIG. 2, comprising FIGS. 2a to 2e . Hepcidin pretreatment mitigateskidney injury following renal IRI. Experimental design and time courseof treatment (a). Plasma creatinine (b) was measured 24 hours after IRIin untreated (◯) or hepcidin injected (50 μg injected 2 hrs: ▪, 8 hrs:Δ, 24 hrs: ∇, or ⋄ [100 μg] mice and compared to sham operated mice (□).Data are represented as mean±SEM. *P<0.05, ***P<0.001, ****P<0.0001(n=4-5 per group). Representative morphology (by H&E staining) ofkidneys after sham operation (c), IRI (d) and hepcidin-IRI (e).Magnification: 10×.

FIG. 3, comprising FIGS. 3a to 3h . Hepcidin reduces apoptosis in thekidneys of mice following IRI and in renal tubular cell line subjectedto hypoxia-reoxygenation regimen.

Hepcidin treatment protects kidney from IRI induced apoptosis. Micereceiving Hepcidin before IRI had a lower transcriptional activation ofpro apoptotic Caspase3 (a) and better preservation of anti apoptoticBcl2 (b). Sham (□), IRI (◯) and Hepcidin+IRI (∇). n=3-5. Data for a andb are represented as mean±SEM of 2^(ΔΔCT) values normalized to GAPDH.TUNEL reactivity was used to assay apoptosis after renal IRI. UntreatedIRI mice showed severe apoptosis in the cells of the corticomedullarregion (c and d), which was reduced by Hepcidin pretreatment (e-f).Representative images from two different experiments with 4-5 mice eachare depicted. Magnification (c and e) 10×, (d and f) 20×. TKPTS cellswere treated with or without 50 μM Hepcidin for 24 hours, beforesubjecting them to 1% Hypoxia for 6 hours followed by 6 hours ofreoxygenation (g-h). Untreated cells cultured under normoxic conditionswere used as controls After 6 hrs of re-oxygenation, cells were stainedfor Annexin V-PI (g-h) and flow cytometry was used to determine theextent of apoptosis. Normoxia (□), Hypoxia (◯), and Hepcidin-Hypoxia (∇)(g-h). Representative of 2 independent experiments. *P<0.05, **P<0.005,***P<0.0001. Data points are plotted as mean±SEM.

FIG. 4, comprising FIGS. 4a to 4c . Hepcidin reduces oxidative stress inthe kidney following IRI and is associated with reduced infiltration ofCD45 positive cells and neutrophils.

Reactivity to 4 hydroxynonenal (4 HNE) was used as a measure ofoxidative stress in the kidney after IRI. Renal IRI resulted ingeneration of ROS mediated oxidative stress as observed by strongreactivity to 4HNE especially in the cortico-medullar region (a) toppanel. Hepcidin treated kidneys had reduced the reactivity to 4 HNEindicative of reduced oxidative stress (a) bottom panel. Representativeimages from two different experiments with 4-5 mice each are depicted.Magnification: 10×. Infiltration of immune cells (CD45+ve) andneutrophils (Ly6G-Ly6c positive cells within CD45−CD11b+ve gates) inkidneys of sham, IRI and Hepcidin treated IRI mice were studied by FACS(b). Representative gates depicting CD45, CD11b, and Ly6GLy6cpopulations are shown in FIG. 4b . The frequency of total CD45 cells(upper panel) as well as neutrophils (lower panel) was reduced inHepcidin treated mice compared to untreated ones (c). Data are from oneof the 3 different experiments with 4-5 mice in each group. Sham (□),IRI (◯) and Hepcidin+IRI (∇). **P<0.001, ***P<0.005, ****P<0.000. Datapoints are plotted as mean±SEM.

FIG. 5, comprising FIGS. 5a to 5d . Hepcidin prevents IRI inducedupregulation of Ferroportin and loss of H Ferritin. Expression offerroportin (a) in the kidney membrane fraction and H-ferritin (c) inthe whole kidney lysates was measured by quantitative Western Blots (aand c). In case of ferroportin blots equal protein loading was confirmedand normalized using Ponceau staining, whereas GAPDH was used to confirmequal loading and normalize H ferritin. Hepcidin prevented the IRIinduced upregulation of (a) Ferroportin, and (c) Loss of H ferritin inthe kidney. Representative blots from 3 independent experiments areshown. Fig b and d: Quantitation of Ferroportin and H ferritin in thekidneys of Sham (□), IRI (◯) and Hepcidin+IRI (∇) mice respectively wascarried out using densitometry software and is expressed as mean±SEM.*P<0.05, **P<0.01.

FIG. 6, comprising FIGS. 6a to 6f . Genetic loss of hepcidin exacerbatesrenal IRI and is mitigated by reconstitution with exogenous Hepcidin.HAMP^(−/−) mice treated with saline or Hepcidin and untreated WT mice(HAMP sufficient) were subjected to mild IRI (24 mins ischemia), andplasma creatinine was measured after 24 hours after IRI (a). The WT micehad a mild but insignificant rise in plasma creatinine (●) compared tosham operated HAMP^(−/−) mice (▪). Untreated HAMP^(−/−)-IRI mice showedhighly significant creatinine (▴) compared to shams. Hepcidinreconstitution rescued the HAMP^(−/−) mice from IRI (▾) and their serumcreatinine values were not significantly different than shams and WTmice. Data are represented as mean±SEM. ****P<0.0001, (n=3-5 per group).Representative morphology of HAMP^(−/−) kidneys (by H&E staining) 24hours after sham operation (b), IRI (c) and hepcidin treatment IRI (d)shows Hepcidin mediated protection of the kidney after IRI.Magnification: 10×. Serum iron was measured by ELISA and normalized tosample volume (e). Hepcidin reconstitution significantly reduced serumiron in HAMP^(−/−)-IRI mice compared to both untreated HAMP^(−/−)-IRIand HAMP^(−/−) sham operated mice. Non-heme iron was measured followingacid digestion of Liver, normalized to tissue weight and expressed asmicro gram per gram tissue (f). Hepcidin treated HAMP^(−/−)-IRI mice hadsignificantly higher non-heme iron compared to both untreatedHAMP^(−/−)-IRI and HAMP^(−/−) sham operated mice. HAMP^(−/−) Sham (●),HAMP^(−/−)-IRI (▪) and HAMP^(−/−)-Hepcidin-IRI (▴). *P<0.05, **P<0.005,***P<0.0005, ****P<0.0001. Data points are plotted as mean±SEM.

FIG. 7, comprising FIGS. 7a to 7g . Hepcidin treatment mitigates theloss of renal H ferritin and is associated with reduced apoptosis.Expression of H-ferritin in the whole kidney lysates was measured byquantitative Western Blots (a). GAPDH was used to confirm equal proteinloading and normalize H ferritin. IRI resulted in a significantreduction in H ferritin in the kidneys of both untreated (◯) andHepcidin treated (∇) HAMP^(−/−) mice as compared to sham (□) HAMP^(−/−)mice (a-b). Hepcidin treated HAMP^(−/−)-IRI mice had significantlyhigher H ferritin levels compared to untreated HAMP^(−/−)-IRI mice.*P<0.05, **P<0.005, ***P<0.0005. Hepcidin reconstitution protectsHAMP^(−/−) kidney from IRI induced apoptosis. Hepcidin treatedHAMP^(−/−)-IRI mice had lower transcriptional activation of proapoptotic Caspase3 (c) and higher level of anti apoptotic Bcl2 (d).HAMP^(−/−) Sham (●), HAMP^(−/−)-IRI (▪) and HAMP^(−/−)-Hepcidin-IRI (▴).n=3-4. *P<0.05, **P<0.005. Data are represented as mean±SEM of 2^(ΔΔCT)normalized to GAPDH. TUNEL reactivity was used to assay apoptosis afterrenal IRI. Sham operated HAMP^(−/−) mice did not show signs of apoptosis(e). Untreated HAMP^(−/−)-IRI mice showed severe apoptosis in the cellsof the corticomedullar region (f), which was markedly reduced in theHepcidin treated HAMP^(−/−)-IRI mice (g). Representative images from twodifferent experiments with 4-5 mice each are depicted. Magnification(10×).

FIG. 8, comprising FIGS. 8a to 8g . Hepcidin reconstitution reduces ROSinduced oxidative stress in the kidneys following IRI under settings ofhepcidin deficiency and results in reduced infiltration of immune cells.Reactivity to 4 hydroxynonenal (4 HNE) was used as an indicator ofoxidative stress in the kidneys of HAMP^(−/−) mice after IRI. Shamoperated HAMP^(−/−) mice did not show reactivity to 4HNE (a). Renal IRIin HAMP^(−/−) mice resulted in ROS mediated oxidative stress as observedby strong reactivity to 4HNE especially in the papilla, deep medulla,and cortico-medullary region (b). Hepcidin treated HAMP^(−/−)-IRIkidneys showed little reactivity to 4 HNE indicative of reducedoxidative stress (c). Representative images from two differentexperiments with 4-5 mice each are depicted. Magnification: 10×.Immunofluorescence labeling of kidneys showed large infiltration ofneutrophils (d, 7/4; green) and CD11b cells (f, CD11b; red) in untreatedHAMP^(−/−) IRI mice. In comparison both, neutrophils (e) as well asCD11b cells (g) are dramatically reduced in Hepcidin treated HAMP^(−/−)IRI mice (arrows). Nuclei were stained with DAPI (blue).

FIG. 9, comprising FIGS. 9a to 9c . Hepcidin treatment prevents renalIRI induced upregulation of splenic ferroportin and increases expressionof cytoprotective H Ferritin. Immunofluorescence labeling of spleen,indicated upregulation of ferroportin (green) in the F4/80 macrophages(red) following renal IRI (a, middle panel). Hepcidin treatment resultedin degradation of ferroportin and could be detected in only fewscattered F4/80 cells in the red pulp region (a, bottom panel). It canbe seen that ferroportin expression is constitutively high is theHAMP^(−/−) mice compared to WT ones (a top panels). Expression ofH-ferritin in the spleen lysates was measured by quantitative WesternBlots using βActin as the loading control. Hepcidin increased splenicH-Ferritin levels in both (b) WT and (c) HAMP^(−/−) after renal IRI.Representative blots from 3 independent experiments are shown. Fig b andc: Quantitation of H-ferritin in the spleens of Sham (●), IRI (▪) andHepcidin-IRI (▴) mice respectively was carried out using densitometrysoftware and is expressed as mean±SEM. *P<0.05 (b), **P<0.005 (c).

FIG. 10, comprising FIGS. 10a to 10c . Hepcidin reduces IRI inducedserum IL6 accumulation. Serum IL6 levels were measured by ELISA.Hepcidin administration significantly reduced IRI induced serum IL6 inboth WT and HAMP^(−/−) mice. In WT mice, IRI resulted in a significantincrease in IL6 compared to sham mice, which was prevented by Hepcidin(a). Similarly, Hepcidin treated HAMP^(−/−)-IRI mice had significantlyreduced serum IL6 compared to untreated HAMP^(−/−)-IRI mice and were notsignificant compared to sham (b). Sham (●), IRI (▪) and Hepcidin-IRI(▴). n=3-5 *P<0.05, **P<0.007.

We treated splenocytes from WT mice with ferrous sulfate and measuredthe IL-6 secretion into supernatants by ELISA. Untreated splenocytes andhepcidin-treated splenocytes were used as controls. There was nosignificant difference in IL-6 levels between the untreated splenocytesand hepcidin-treated cells (FIG. 10c ). However treatment with ironsignificantly increased splenocytes IL-6 secretion compared to bothuntreated and hepcidin-treated cells (FIG. 10c , p<0.005).

FIG. 11. Pooled minihepcidin (PR-73) data showing protection in AKI.Animals were subjected to IRI with or without treatment with thehepcidin agonist at 100 or 200 nmol. The effects at 200 nmol arestatistically significant relative to the other groups.

FIG. 12, comprising FIGS. 12a to 12d (also referred to as SupportingFIG. 1). Representative morphology (by H&E staining) of kidneys aftersham operation (a), IRI (b), and hepcidin-IRI (c). Magnification: 10×(a, b, c), Original magnification 20× (insets). Relative NGAL expressionin the kidneys of Sham (□), IRI (◯) and Hepcidin-IRI (∇) normalized toGAPDH (d). Data are represented as mean±SEM. ***P<0.001, ****P<0.0001(n=4-5 per group).

FIG. 13, comprising FIGS. 13a to 13b (also referred to as SupportingFIG. 2). High magnification images of 4HNE staining. Untreated WT IRImice showed strong reactivity to 4HNE (a), which was markedly reduced inthe Hepcidin treated WT-IRI mice (b). Representative images from twodifferent experiments with 4-5 mice each are depicted. Magnification20×.

FIG. 14, comprising FIGS. 14a to 14d (also referred to as SupportingFIG. 3) Representative morphology of HAMP^(−/−) kidneys (by H&Estaining) 24 hours after sham operation (a), IRI (b) and hepcidintreatment IRI (c) shows Hepcidin mediated protection of the kidney afterIRI. Magnification: 10×.

Relative NGAL expression in the kidneys of HAMP^(−/−) Sham (●),HAMP^(−/−)-IRI (▪) and HAMP^(−/−)-Hepcidin-IRI (▴), normalized to GAPDH(d). Data are represented as mean±SEM. *P<0.05, **P<0.005 (n=3-4 pergroup).

FIG. 15, comprising FIGS. 15a to 15c (also referred to as SupportingFIG. 4). High magnification images of TUNEL staining. Sham operatedHAMP^(−/−) mice did not show signs of apoptosis (a). UntreatedHAMP^(−/−)-IRI mice showed severe apoptosis in the cells of thecorticomedullar region (b), which was markedly reduced in the Hepcidintreated HAMP^(−/−)-IRI mice (c). Representative images from twodifferent experiments with 4-5 mice each are depicted. Magnification20×, Scale bar: 100 μm.

DETAILED DESCRIPTION Abbreviation and Acronyms

4-HNE—4 hydroxynonenal

a.a.—amino acid

AI—anemia of inflammation

AKI—acute kidney injury

ASO—antisense oligonucleotide

ATN—acute tubular necrosis

BMP—bone morphogenetic protein

EGF—epidermal growth factor

FAC—ferric ammonium chloride

FCS—fetal calf serum

H&E—hematoxylin and eosin

HAMP—hepcidin

HGF—hepatocyte growth factor

HJV—hemojuvelin

IL-6—interleukin-6

IRI—ischemia reperfusion injury

kg—kilogram

mg—milligram

OH—hydroxyl

PG-APS—streptococcal peptidoglycan-polysaccharide

PLP—paraformaldehyde/lysine/periodate

ROS—reactive oxygen species

sHJV.Fc—Soluble HJV-Fc fusion protein

SOD—superoxide dismutase

TEC—tubular epithelial cell

TfR1—transferrin receptor 1

TfR2—transferrin receptor 2

TMPRSS6—transmembrane serine protease, also known as matriptase-2

TUNEL—terminal deoxynucleotidyl transferase-mediateddigoxigenin-deoxyuridine nick-end labeling

wt.—weight

WT—wild type

Definitions

In describing and claiming the invention, the following terminology willbe used in accordance with the definitions set forth below.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

The term “about,” as used herein, means approximately, in the region of,roughly, or around. When the term “about” is used in conjunction with anumerical range, it modifies that range by extending the boundariesabove and below the numerical values set forth. In general, the term“about” is used herein to modify a numerical value above and below thestated value by a variance of 10%. In one aspect, the term “about” meansplus or minus 20% of the numerical value of the number with which it isbeing used. Therefore, about 50% means in the range of 45%-55%.Numerical ranges recited herein by endpoints include all numbers andfractions subsumed within that range (e.g. 1 to 5 includes 1, 1.5, 2,2.75, 3, 3.90, 4, and 5). It is also to be understood that all numbersand fractions thereof are presumed to be modified by the term “about.”

The terms “additional therapeutically active compound” or “additionaltherapeutic agent”, as used in the context of the present invention,refers to the use or administration of a compound for an additionaltherapeutic use for a particular injury, disease, or disorder beingtreated. Such a compound, for example, could include one being used totreat an unrelated disease or disorder, or a disease or disorder whichmay not be responsive to the primary treatment for the injury, diseaseor disorder being treated.

As used herein, the term “adjuvant” refers to a substance that elicitsan enhanced immune response when used in combination with a specificantigen.

As use herein, the terms “administration of” and or “administering” acompound should be understood to mean providing a compound of theinvention or a prodrug of a compound of the invention to a subject inneed of treatment.

As used herein, the term “aerosol” refers to suspension in the air. Inparticular, aerosol refers to the particlization or atomization of aformulation of the invention and its suspension in the air.

As used herein, an “agonist” is a composition of matter which, whenadministered to a mammal such as a human, enhances or extends abiological activity attributable to the level or presence of a targetcompound or molecule of interest in the mammal.

The term “alterations in peptide structure” as used herein refers tochanges including, but not limited to, changes in sequence, andpost-translational modification.

An “antagonist” is a composition of matter which when administered to amammal such as a human, inhibits a biological activity attributable tothe level or presence of a compound or molecule of interest in themammal.

As used herein, “alleviating a disease or disorder symptom,” meansreducing the severity of the symptom or the frequency with which such asymptom is experienced by a patient, or both.

As used herein, amino acids are represented by the full name thereof, bythe three letter code corresponding thereto, or by the one-letter codecorresponding thereto, as indicated in the following table:

Full Name Three-Letter Code One-Letter Code Aspartic Acid Asp D GlutamicAcid Glu E Lysine Lys K Arginine Arg R Histidine His H Tyrosine Tyr YCysteine Cys C Asparagine Asn N Glutamine Gln Q Serine Ser S ThreonineThr T Glycine Gly G Alanine Ala A Valine Val V Leucine Leu L IsoleucineIle I Methionine Met M Proline Pro P Phenylalanine Phe F Tryptophan TrpW

The term “amino acid” is used interchangeably with “amino acid residue,”and may refer to a free amino acid and to an amino acid residue of apeptide. It will be apparent from the context in which the term is usedwhether it refers to a free amino acid or a residue of a peptide.

Amino acids have the following general structure:

Amino acids may be classified into seven groups on the basis of the sidechain R: (1) aliphatic side chains, (2) side chains containing ahydroxylic (OH) group, (3) side chains containing sulfur atoms, (4) sidechains containing an acidic or amide group, (5) side chains containing abasic group, (6) side chains containing an aromatic ring, and (7)proline, an imino acid in which the side chain is fused to the aminogroup.

The nomenclature used to describe the peptide compounds of the presentinvention follows the conventional practice wherein the amino group ispresented to the left and the carboxy group to the right of each aminoacid residue. In the formulae representing selected specific embodimentsof the present invention, the amino- and carboxy-terminal groups,although not specifically shown, will be understood to be in the formthey would assume at physiologic pH values, unless otherwise specified.

The term “basic” or “positively charged” amino acid as used herein,refers to amino acids in which the R groups have a net positive chargeat pH 7.0, and include, but are not limited to, the standard amino acidslysine, arginine, and histidine.

The term “an effective amount of hepcidin” as used herein refers to theuse of a full-length hepcidin or a biologically active fragment orhomolog thereof that is effective for the use described herein.

As used herein, an “analog” of a chemical compound is a compound that,by way of example, resembles another in structure but is not necessarilyan isomer (e.g., 5-fluorouracil is an analog of thymine).

The term “antibody,” as used herein, refers to an immunoglobulinmolecule which is able to specifically bind to a specific epitope on anantigen. Antibodies can be intact immunoglobulins derived from naturalsources or from recombinant sources and can be immunoreactive portionsof intact immunoglobulins. Antibodies are typically tetramers ofimmunoglobulin molecules. The antibodies in the present invention mayexist in a variety of forms including, for example, polyclonalantibodies, monoclonal antibodies, Fv, Fab and F(ab)₂, as well as singlechain antibodies and humanized antibodies.

The term “antibody” refers to polyclonal and monoclonal antibodies andderivatives thereof (including chimeric, synthesized, humanized andhuman antibodies), including an entire immunoglobulin or antibody or anyfunctional fragment of an immunoglobulin molecule which binds to thetarget antigen and or combinations thereof. Examples of such functionalentities include complete antibody molecules, antibody fragments, suchas F_(v), single chain F_(v), complementarity determining regions(CDRs), V_(L) (light chain variable region), V_(H) (heavy chain variableregion), Fab, F(ab′)₂ and any combination of those or any otherfunctional portion of an immunoglobulin peptide capable of binding totarget antigen.

Antibodies exist, e.g., as intact immunoglobulins or as a number of wellcharacterized fragments produced by digestion with various peptidases.Thus, for example, pepsin digests an antibody below the disulfidelinkages in the hinge region to produce F(ab′)₂ a dimer of Fab whichitself is a light chain joined to V_(H)-C_(H1) by a disulfide bond. TheF(ab′)₂ may be reduced under mild conditions to break the disulfidelinkage in the hinge region, thereby converting the F(ab′)₂ dimer intoan Fab₁ monomer. The Fab₁ monomer is essentially an Fab with part of thehinge region (see, FUNDAMENTAL IMMUNOLOGY, 3RD ED., W. E. Paul, ed.,Raven Press, N.Y. (1993)). While various antibody fragments are definedin terms of the digestion of an intact antibody, one of skill willappreciate that such fragments may be synthesized de novo eitherchemically or by utilizing recombinant DNA methodology. Thus, the termantibody, as used herein, also includes antibody fragments eitherproduced by the modification of whole antibodies or those synthesized denovo using recombinant DNA methodologies.

An “antibody heavy chain,” as used herein, refers to the larger of thetwo types of polypeptide chains present in all antibody molecules.

An “antibody light chain,” as used herein, refers to the smaller of thetwo types of polypeptide chains present in all antibody molecules.

The term “single chain antibody” refers to an antibody wherein thegenetic information encoding the functional fragments of the antibodyare located in a single contiguous length of DNA. For a thoroughdescription of single chain antibodies, see Bire, et al., Science242:423 (1988) and Huston, et al., Proc. Nat'l Acad. Sci. USA 85:5879(1988).

The term “humanized” refers to an antibody wherein the constant regionshave at least about 80% or greater homology to human immunoglobulin.Additionally, some of the nonhuman, such as murine, variable regionamino acid residues can be modified to contain amino acid residues ofhuman origin.

Humanized antibodies have been referred to as “reshaped” antibodies.Manipulation of the complementarity-determining regions (CDR) is a wayof achieving humanized antibodies. See, for example, Jones, et al.,Nature 321:522 (1988) and Riechmann, et al., Nature 332:323 (1988), bothof which are incorporated by reference herein. For a review articleconcerning humanized antibodies, see Winter & Milstein, Nature 349:293(1991), incorporated by reference herein.

By the term “synthetic antibody” as used herein, is meant an antibodywhich is generated using recombinant DNA technology, such as, forexample, an antibody expressed by a bacteriophage as described herein.The term should also be construed to mean an antibody which has beengenerated by the synthesis of a DNA molecule encoding the antibody andwhich DNA molecule expresses an antibody protein, or an amino acidsequence specifying the antibody, wherein the DNA or amino acid sequencehas been obtained using synthetic DNA or amino acid sequence technologywhich is available and well known in the art.

The term “antigen” as used herein is defined as a molecule that provokesan immune response. This immune response may involve either antibodyproduction, or the activation of specific immunologically-competentcells, or both. An antigen can be derived from organisms, subunits ofproteins/antigens, killed or inactivated whole cells or lysates.

The term “antimicrobial agents” as used herein refers to anynaturally-occurring, synthetic, or semi-synthetic compound orcomposition or mixture thereof, which is safe for human or animal use aspracticed in the methods of this invention, and is effective in killingor substantially inhibiting the growth of microbes. “Antimicrobial” asused herein, includes antibacterial, antifungal, and antiviral agents.

As used herein, the term “antisense oligonucleotide” or antisensenucleic acid means a nucleic acid polymer, at least a portion of whichis complementary to a nucleic acid which is present in a normal cell orin an affected cell. “Antisense” refers particularly to the nucleic acidsequence of the non-coding strand of a double stranded DNA moleculeencoding a protein, or to a sequence which is substantially homologousto the non-coding strand. As defined herein, an antisense sequence iscomplementary to the sequence of a double stranded DNA molecule encodinga protein. It is not necessary that the antisense sequence becomplementary solely to the coding portion of the coding strand of theDNA molecule. The antisense sequence may be complementary to regulatorysequences specified on the coding strand of a DNA molecule encoding aprotein, which regulatory sequences control expression of the codingsequences. The antisense oligonucleotides of the invention include, butare not limited to, phosphorothioate oligonucleotides and othermodifications of oligonucleotides.

An “aptamer” is a compound that is selected in vitro to bindpreferentially to another compound (for example, the identified proteinsherein). Often, aptamers are nucleic acids or peptides because randomsequences can be readily generated from nucleotides or amino acids (bothnaturally occurring or synthetically made) in large numbers but ofcourse they need not be limited to these.

The term “binding” refers to the adherence of molecules to one another,such as, but not limited to, enzymes to substrates, ligands toreceptors, antibodies to antigens, DNA binding domains of proteins toDNA, and DNA or RNA strands to complementary strands.

“Binding partner,” as used herein, refers to a molecule capable ofbinding to another molecule.

The term “biocompatible”, as used herein, refers to a material that doesnot elicit a substantial detrimental response in the host.

As used herein, the term “biologically active fragments” or “bioactivefragment” of the polypeptides encompasses natural or synthetic portionsof the full-length protein that are capable of specific binding to theirnatural ligand or of performing the function of the protein.

The term “biological sample,” as used herein, refers to samples obtainedfrom a subject, including, but not limited to, sputum, mucus, phlegm,tissues, biopsies, cerebrospinal fluid, blood, serum, plasma, otherblood components, gastric aspirates, throat swabs, pleural effusion,peritoneal fluid, follicular fluid, ascites, skin, hair, tissue, blood,plasma, cells, saliva, sweat, tears, semen, stools, Pap smears, andurine. One of skill in the art will understand the type of sampleneeded.

A “biomarker” or “marker” is a specific biochemical in the body whichhas a particular molecular feature that makes it useful for measuringthe progress of disease or the effects of treatment, or for measuring aprocess of interest.

The term “cancer”, as used herein, is defined as proliferation of cellswhose unique trait (loss of normal controls) results in unregulatedgrowth, lack of differentiation, local tissue invasion, and metastasis.Examples include but are not limited to, melanoma, breast cancer,prostate cancer, ovarian cancer, uterine cancer, cervical cancer, skincancer, pancreatic cancer, colorectal cancer, renal cancer and lungcancer.

As used herein, the term “carrier molecule” refers to any molecule thatis chemically conjugated to a molecule of interest.

The term “cell surface protein” means a protein found where at leastpart of the protein is exposed at the outer aspect of the cell membrane.Examples include growth factor receptors.

As used herein, the term “chemically conjugated,” or “conjugatingchemically” refers to linking the antigen to the carrier molecule. Thislinking can occur on the genetic level using recombinant technology,wherein a hybrid protein may be produced containing the amino acidsequences, or portions thereof, of both the antigen and the carriermolecule. This hybrid protein is produced by an oligonucleotide sequenceencoding both the antigen and the carrier molecule, or portions thereof.This linking also includes covalent bonds created between the antigenand the carrier protein using other chemical reactions, such as, but notlimited to glutaraldehyde reactions. Covalent bonds may also be createdusing a third molecule bridging the antigen to the carrier molecule.These cross-linkers are able to react with groups, such as but notlimited to, primary amines, sulfhydryls, carbonyls, carbohydrates, orcarboxylic acids, on the antigen and the carrier molecule. Chemicalconjugation also includes non-covalent linkage between the antigen andthe carrier molecule.

A “coding region” of a gene consists of the nucleotide residues of thecoding strand of the gene and the nucleotides of the non-coding strandof the gene which are homologous with or complementary to, respectively,the coding region of an mRNA molecule which is produced by transcriptionof the gene.

The term “competitive sequence” refers to a peptide or a modification,fragment, derivative, or homolog thereof that competes with anotherpeptide for its cognate binding site.

“Complementary” as used herein refers to the broad concept of subunitsequence complementarity between two nucleic acids, e.g., two DNAmolecules. When a nucleotide position in both of the molecules isoccupied by nucleotides normally capable of base pairing with eachother, then the nucleic acids are considered to be complementary to eachother at this position. Thus, two nucleic acids are complementary toeach other when a substantial number (at least 50%) of correspondingpositions in each of the molecules are occupied by nucleotides whichnormally base pair with each other (e.g., A:T and G:C nucleotide pairs).Thus, it is known that an adenine residue of a first nucleic acid regionis capable of forming specific hydrogen bonds (“base pairing”) with aresidue of a second nucleic acid region which is antiparallel to thefirst region if the residue is thymine or uracil. Similarly, it is knownthat a cytosine residue of a first nucleic acid strand is capable ofbase pairing with a residue of a second nucleic acid strand which isantiparallel to the first strand if the residue is guanine. A firstregion of a nucleic acid is complementary to a second region of the sameor a different nucleic acid if, when the two regions are arranged in anantiparallel fashion, at least one nucleotide residue of the firstregion is capable of base pairing with a residue of the second region.Preferably, the first region comprises a first portion and the secondregion comprises a second portion, whereby, when the first and secondportions are arranged in an antiparallel fashion, at least about 50%,and preferably at least about 75%, at least about 90%, or at least about95% of the nucleotide residues of the first portion are capable of basepairing with nucleotide residues in the second portion. More preferably,all nucleotide residues of the first portion are capable of base pairingwith nucleotide residues in the second portion.

A “compound,” as used herein, refers to any type of substance or agentthat is commonly considered a drug, or a candidate for use as a drug, aswell as combinations and mixtures of the above.

A “computer-readable medium” is an information storage medium that canbe accessed by a computer using a commercially available or custom-madeinterface. Exemplary compute-readable media include memory (e.g., RAM,ROM, flash memory, etc.), optical storage media (e.g., CD-ROM), magneticstorage media (e.g., computer hard drives, floppy disks, etc.), punchcards, or other commercially available media. Information may betransferred between a system of interest and a medium, betweencomputers, or between computers and the computer-readable medium forstorage or access of stored information. Such transmission can beelectrical, or by other available methods, such as IR links, wirelessconnections, etc.

As used herein, the term “conservative amino acid substitution” isdefined herein as an amino acid exchange within one of the followingfive groups:

I. Small aliphatic, nonpolar or slightly polar residues:

-   -   Ala, Ser, Thr, Pro, Gly;

II. Polar, negatively charged residues and their amides:

-   -   Asp, Asn, Glu, Gln;

III. Polar, positively charged residues:

-   -   His, Arg, Lys;

IV. Large, aliphatic, nonpolar residues:

-   -   Met Leu, Ile, Val, Cys

V. Large, aromatic residues:

-   -   Phe, Tyr, Trp

A “control” cell is a cell having the same cell type as a test cell. Thecontrol cell may, for example, be examined at precisely or nearly thesame time the test cell is examined. The control cell may also, forexample, be examined at a time distant from the time at which the testcell is examined, and the results of the examination of the control cellmay be recorded so that the recorded results may be compared withresults obtained by examination of a test cell.

A “test” cell is a cell being examined.

As used herein, a “derivative” of a compound refers to a chemicalcompound that may be produced from another compound of similar structurein one or more steps, as in replacement of H by an alkyl, acyl, or aminogroup.

As used herein “detachment-induced chemoresistance” refers to the changein cancer cells that occurs when they become resistant to chemotherapyfollowing a change in their interactions with the extracellular matrix.

The use of the word “detect” and its grammatical variants refers tomeasurement of the species without quantification, whereas use of theword “determine” or “measure” with their grammatical variants are meantto refer to measurement of the species with quantification. The terms“detect” and “identify” are used interchangeably herein.

As used herein, a “detectable marker” or a “reporter molecule” is anatom or a molecule that permits the specific detection of a compoundcomprising the marker in the presence of similar compounds without amarker. Detectable markers or reporter molecules include, e.g.,radioactive isotopes, antigenic determinants, enzymes, nucleic acidsavailable for hybridization, chromophores, fluorophores,chemiluminescent molecules, electrochemically detectable molecules, andmolecules that provide for altered fluorescence-polarization or alteredlight-scattering.

As used herein, in one embodiment, the term “diagnosis” refers todetecting aberrant ALCAM expression due to cancers expressing ALCAM. Inany method of diagnosis exist false positives and false negatives. Anyone method of diagnosis does not provide 100% accuracy.

A “disease” is a state of health of an animal wherein the animal cannotmaintain homeostasis, and wherein if the disease is not ameliorated thenthe animal's health continues to deteriorate.

In contrast, a “disorder” in an animal is a state of health in which theanimal is able to maintain homeostasis, but in which the animal's stateof health is less favorable than it would be in the absence of thedisorder. Left untreated, a disorder does not necessarily cause afurther decrease in the animal's state of health.

As used herein, the term “domain” refers to a part of a molecule orstructure that shares common physicochemical features, such as, but notlimited to, hydrophobic, polar, globular and helical domains orproperties such as ligand binding, signal transduction, cell penetrationand the like. Specific examples of binding domains include, but are notlimited to, DNA binding domains and ATP binding domains.

As used herein, an “effective amount” or “therapeutically effectiveamount” means an amount sufficient to produce a selected effect, such asalleviating symptoms of a disease or disorder. In the context ofadministering compounds in the form of a combination, such as multiplecompounds, the amount of each compound, when administered in combinationwith another compound(s), may be different from when that compound isadministered alone. Thus, an effective amount of a combination ofcompounds refers collectively to the combination as a whole, althoughthe actual amounts of each compound may vary. The term “more effective”means that the selected effect is alleviated to a greater extent by onetreatment relative to the second treatment to which it is beingcompared.

As used herein, the term “effector domain” refers to a domain capable ofdirectly interacting with an effector molecule, chemical, or structurein the cytoplasm which is capable of regulating a biochemical pathway.

The term “elixir,” as used herein, refers in general to a clear,sweetened, alcohol-containing, usually hydroalcoholic liquid containingflavoring substances and sometimes active medicinal agents.

“Encoding” refers to the inherent property of specific sequences ofnucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, toserve as templates for synthesis of other polymers and macromolecules inbiological processes having either a defined sequence of nucleotides(i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and thebiological properties resulting therefrom. Thus, a gene encodes aprotein if transcription and translation of mRNA corresponding to thatgene produces the protein in a cell or other biological system. Both thecoding strand, the nucleotide sequence of which is identical to the mRNAsequence and is usually provided in sequence listings, and thenon-coding strand, used as the template for transcription of a gene orcDNA, can be referred to as encoding the protein or other product ofthat gene or cDNA.

An “enhancer” is a DNA regulatory element that can increase theefficiency of transcription, regardless of the distance or orientationof the enhancer relative to the start site of transcription.

The term “epitope” as used herein is defined as small chemical groups onthe antigen molecule that can elicit and react with an antibody. Anantigen can have one or more epitopes. Most antigens have many epitopes;i.e., they are multivalent. In general, an epitope is roughly five aminoacids or sugars in size. One skilled in the art understands thatgenerally the overall three-dimensional structure, rather than thespecific linear sequence of the molecule, is the main criterion ofantigenic specificity.

As used herein, an “essentially pure” preparation of a particularprotein or peptide is a preparation wherein at least about 95%, andpreferably at least about 99%, by weight, of the protein or peptide inthe preparation is the particular protein or peptide.

A “fragment” or “segment” is a portion of an amino acid sequence,comprising at least one amino acid, or a portion of a nucleic acidsequence comprising at least one nucleotide. The terms “fragment” and“segment” are used interchangeably herein.

As used herein, the term “fragment,” as applied to a protein or peptide,can ordinarily be at least about 3-15 amino acids in length, at leastabout 15-25 amino acids, at least about 25-50 amino acids in length, atleast about 50-75 amino acids in length, at least about 75-100 aminoacids in length, and greater than 100 amino acids in length.

As used herein, the term “fragment” as applied to a nucleic acid, mayordinarily be at least about 20 nucleotides in length, typically, atleast about 50 nucleotides, more typically, from about 50 to about 100nucleotides, preferably, at least about 100 to about 200 nucleotides,even more preferably, at least about 200 nucleotides to about 300nucleotides, yet even more preferably, at least about 300 to about 350,even more preferably, at least about 350 nucleotides to about 500nucleotides, yet even more preferably, at least about 500 to about 600,even more preferably, at least about 600 nucleotides to about 620nucleotides, yet even more preferably, at least about 620 to about 650,and most preferably, the nucleic acid fragment will be greater thanabout 650 nucleotides in length.

As used herein, a “functional” biological molecule is a biologicalmolecule in a form in which it exhibits a property by which it ischaracterized. A functional enzyme, for example, is one which exhibitsthe characteristic catalytic activity by which the enzyme ischaracterized.

“Homologous” as used herein, refers to the subunit sequence similaritybetween two polymeric molecules, e.g., between two nucleic acidmolecules, e.g., two DNA molecules or two RNA molecules, or between twopolypeptide molecules. When a subunit position in both of the twomolecules is occupied by the same monomeric subunit, e.g., if a positionin each of two DNA molecules is occupied by adenine, then they arehomologous at that position. The homology between two sequences is adirect function of the number of matching or homologous positions, e.g.,if half (e.g., five positions in a polymer ten subunits in length) ofthe positions in two compound sequences are homologous then the twosequences are 50% homologous, if 90% of the positions, e.g., 9 of 10,are matched or homologous, the two sequences share 90% homology. By wayof example, the DNA sequences 3′ATTGCC5′ and 3′TATGGC share 50%homology.

As used herein, “homology” is used synonymously with “identity.”

The determination of percent identity between two nucleotide or aminoacid sequences can be accomplished using a mathematical algorithm. Forexample, a mathematical algorithm useful for comparing two sequences isthe algorithm of Karlin and Altschul (1990, Proc. Natl. Acad. Sci. USA87:2264-2268), modified as in Karlin and Altschul (1993, Proc. Natl.Acad. Sci. USA 90:5873-5877). This algorithm is incorporated into theNBLAST and XBLAST programs of Altschul, et al. (1990, J. Mol. Biol.215:403-410), and can be accessed, for example at the National Centerfor Biotechnology Information (NCBI) world wide web site having theuniversal resource locator using the BLAST tool at the NCBI website.BLAST nucleotide searches can be performed with the NBLAST program(designated “blastn” at the NCBI web site), using the followingparameters: gap penalty=5; gap extension penalty=2; mismatch penalty=3;match reward=1; expectation value 10.0; and word size=11 to obtainnucleotide sequences homologous to a nucleic acid described herein.BLAST protein searches can be performed with the XBLAST program(designated “blastn” at the NCBI web site) or the NCBI “blastp” program,using the following parameters: expectation value 10.0, BLOSUM62 scoringmatrix to obtain amino acid sequences homologous to a protein moleculedescribed herein. To obtain gapped alignments for comparison purposes,Gapped BLAST can be utilized as described in Altschul et al. (1997,Nucleic Acids Res. 25:3389-3402). Alternatively, PSI-Blast or PHI-Blastcan be used to perform an iterated search which detects distantrelationships between molecules (Id.) and relationships betweenmolecules which share a common pattern. When utilizing BLAST, GappedBLAST, PSI-Blast, and PHI-Blast programs, the default parameters of therespective programs (e.g., XBLAST and NBLAST) can be used.

The percent identity between two sequences can be determined usingtechniques similar to those described above, with or without allowinggaps. In calculating percent identity, typically exact matches arecounted.

As used herein, the term “hybridization” is used in reference to thepairing of complementary nucleic acids. Hybridization and the strengthof hybridization (i.e., the strength of the association between thenucleic acids) is impacted by such factors as the degree ofcomplementarity between the nucleic acids, stringency of the conditionsinvolved, the length of the formed hybrid, and the G:C ratio within thenucleic acids.

As used herein, the term “inhaler” refers both to devices for nasal andpulmonary administration of a drug, e.g., in solution, powder and thelike. For example, the term “inhaler” is intended to encompass apropellant driven inhaler, such as is used to administer antihistaminefor acute asthma attacks, and plastic spray bottles, such as are used toadminister decongestants.

The term “inhibit,” as used herein, refers to the ability of a compound,agent, or method to reduce or impede a described function, level,activity, rate, etc., based on the context in which the term “inhibit”is used. Preferably, inhibition is by at least 10%, more preferably byat least 25%, even more preferably by at least 50%, and most preferably,the function is inhibited by at least 75%. The term “inhibit” is usedinterchangeably with “reduce” and “block.”

The term “inhibit a complex,” as used herein, refers to inhibiting theformation of a complex or interaction of two or more proteins, as wellas inhibiting the function or activity of the complex. The term alsoencompasses disrupting a formed complex. However, the term does notimply that each and every one of these functions must be inhibited atthe same time.

The term “inhibit a protein,” as used herein, refers to any method ortechnique which inhibits protein synthesis, levels, activity, orfunction, as well as methods of inhibiting the induction or stimulationof synthesis, levels, activity, or function of the protein of interest.The term also refers to any metabolic or regulatory pathway which canregulate the synthesis, levels, activity, or function of the protein ofinterest. The term includes binding with other molecules and complexformation. Therefore, the term “protein inhibitor” refers to any agentor compound, the application of which results in the inhibition ofprotein function or protein pathway function. However, the term does notimply that each and every one of these functions must be inhibited atthe same time.

As used herein “injecting or applying” includes administration of acompound of the invention by any number of routes and means including,but not limited to, topical, oral, buccal, intravenous, intramuscular,intra arterial, intramedullary, intrathecal, intraventricular,transdermal, subcutaneous, intraperitoneal, intranasal, enteral,topical, sublingual, vaginal, ophthalmic, pulmonary, or rectal means.Compounds or agents of the invention can be administered to a subject bythese means when appropriate.

As used herein, an “instructional material” includes a publication, arecording, a diagram, or any other medium of expression which can beused to communicate the usefulness of the peptide of the invention inthe kit for effecting alleviation of the various diseases or disordersrecited herein. Optionally, or alternately, the instructional materialmay describe one or more methods of alleviating the diseases ordisorders in a cell or a tissue of a mammal. The instructional materialof the kit of the invention may, for example, be affixed to a containerwhich contains the identified compound invention or be shipped togetherwith a container which contains the identified compound. Alternatively,the instructional material may be shipped separately from the containerwith the intention that the instructional material and the compound beused cooperatively by the recipient.

An “isolated nucleic acid” refers to a nucleic acid segment or fragmentwhich has been separated from sequences which flank it in a naturallyoccurring state, e.g., a DNA fragment which has been removed from thesequences which are normally adjacent to the fragment, e.g., thesequences adjacent to the fragment in a genome in which it naturallyoccurs. The term also applies to nucleic acids which have beensubstantially purified from other components which naturally accompanythe nucleic acid, e.g., RNA or DNA or proteins, which naturallyaccompany it in the cell. The term therefore includes, for example, arecombinant DNA which is incorporated into a vector, into anautonomously replicating plasmid or virus, or into the genomic DNA of aprokaryote or eukaryote, or which exists as a separate molecule (e.g.,as a cDNA or a genomic or cDNA fragment produced by PCR or restrictionenzyme digestion) independent of other sequences. It also includes arecombinant DNA which is part of a hybrid gene encoding additionalpolypeptide sequence.

A “ligand” is a compound that specifically binds to a target receptor.

A “receptor” is a compound that specifically binds to a ligand.

A ligand or a receptor (e.g., an antibody) “specifically binds to” or“is specifically immunoreactive with” a compound when the ligand orreceptor functions in a binding reaction which is determinative of thepresence of the compound in a sample of heterogeneous compounds. Thus,under designated assay (e.g., immunoassay) conditions, the ligand orreceptor binds preferentially to a particular compound and does not bindin a significant amount to other compounds present in the sample. Forexample, a polynucleotide specifically binds under hybridizationconditions to a compound polynucleotide comprising a complementarysequence; an antibody specifically binds under immunoassay conditions toan antigen bearing an epitope against which the antibody was raised. Avariety of immunoassay formats may be used to select antibodiesspecifically immunoreactive with a particular protein. For example,solid-phase ELISA immunoassays are routinely used to select monoclonalantibodies specifically immunoreactive with a protein. See Harlow andLane (1988, Antibodies, A Laboratory Manual, Cold Spring HarborPublications, New York) for a description of immunoassay formats andconditions that can be used to determine specific immunoreactivity.

As used herein, the term “linkage” refers to a connection between twogroups. The connection can be either covalent or non-covalent, includingbut not limited to ionic bonds, hydrogen bonding, andhydrophobic/hydrophilic interactions.

As used herein, the term “linker” refers to a molecule that joins twoother molecules either covalently or noncovalently, e.g., through ionicor hydrogen bonds or van der Waals interactions, e.g., a nucleic acidmolecule that hybridizes to one complementary sequence at the 5′ end andto another complementary sequence at the 3′ end, thus joining twonon-complementary sequences.

“Malexpression” of a gene means expression of a gene in a cell of apatient afflicted with a disease or disorder, wherein the level ofexpression (including non-expression), the portion of the geneexpressed, or the timing of the expression of the gene with regard tothe cell cycle, differs from expression of the same gene in a cell of apatient not afflicted with the disease or disorder. It is understoodthat malexpression may cause or contribute to the disease or disorder,be a symptom of the disease or disorder, or both.

The term “measuring the level of expression” or “determining the levelof expression” as used herein refers to any measure or assay which canbe used to correlate the results of the assay with the level ofexpression of a gene or protein of interest. Such assays includemeasuring the level of mRNA, protein levels, etc. and can be performedby assays such as northern and western blot analyses, binding assays,immunoblots, etc. The level of expression can include rates ofexpression and can be measured in terms of the actual amount of an mRNAor protein present. Such assays are coupled with processes or systems tostore and process information and to help quantify levels, signals, etc.and to digitize the information for use in comparing levels.

The term “nasal administration” in all its grammatical forms refers toadministration of at least one compound of the invention through thenasal mucous membrane to the bloodstream for systemic delivery of atleast one compound of the invention. The advantages of nasaladministration for delivery are that it does not require injection usinga syringe and needle, it avoids necrosis that can accompanyintramuscular administration of drugs, and trans-mucosal administrationof a drug is highly amenable to self-administration.

The term “nucleic acid” typically refers to large polynucleotides. By“nucleic acid” is meant any nucleic acid, whether composed ofdeoxyribonucleosides or ribonucleosides, and whether composed ofphosphodiester linkages or modified linkages such as phosphotriester,phosphoramidate, siloxane, carbonate, carboxymethylester, acetamidate,carbamate, thioether, bridged phosphoramidate, bridged methylenephosphonate, bridged phosphoramidate, bridged phosphoramidate, bridgedmethylene phosphonate, phosphorothioate, methylphosphonate,phosphorodithioate, bridged phosphorothioate or sulfone linkages, andcombinations of such linkages. The term nucleic acid also specificallyincludes nucleic acids composed of bases other than the fivebiologically occurring bases (adenine, guanine, thymine, cytosine anduracil).

As used herein, the term “nucleic acid” encompasses RNA as well assingle and double-stranded DNA and cDNA. Furthermore, the terms,“nucleic acid,” “DNA,” “RNA” and similar terms also include nucleic acidanalogs, i.e. analogs having other than a phosphodiester backbone. Forexample, the so-called “peptide nucleic acids,” which are known in theart and have peptide bonds instead of phosphodiester bonds in thebackbone, are considered within the scope of the present invention. By“nucleic acid” is meant any nucleic acid, whether composed ofdeoxyribonucleosides or ribonucleosides, and whether composed ofphosphodiester linkages or modified linkages such as phosphotriester,phosphoramidate, siloxane, carbonate, carboxymethylester, acetamidate,carbamate, thioether, bridged phosphoramidate, bridged methylenephosphonate, bridged phosphoramidate, bridged phosphoramidate, bridgedmethylene phosphonate, phosphorothioate, methylphosphonate,phosphorodithioate, bridged phosphorothioate or sulfone linkages, andcombinations of such linkages. The term nucleic acid also specificallyincludes nucleic acids composed of bases other than the fivebiologically occurring bases (adenine, guanine, thymine, cytosine, anduracil). Conventional notation is used herein to describe polynucleotidesequences: the left-hand end of a single-stranded polynucleotidesequence is the 5′-end; the left-hand direction of a double-strandedpolynucleotide sequence is referred to as the 5′-direction. Thedirection of 5′ to 3′ addition of nucleotides to nascent RNA transcriptsis referred to as the transcription direction. The DNA strand having thesame sequence as an mRNA is referred to as the “coding strand”;sequences on the DNA strand which are located 5′ to a reference point onthe DNA are referred to as “upstream sequences”; sequences on the DNAstrand which are 3′ to a reference point on the DNA are referred to as“downstream sequences.”

The term “nucleic acid construct,” as used herein, encompasses DNA andRNA sequences encoding the particular gene or gene fragment desired,whether obtained by genomic or synthetic methods.

Unless otherwise specified, a “nucleotide sequence encoding an aminoacid sequence” includes all nucleotide sequences that are degenerateversions of each other and that encode the same amino acid sequence.Nucleotide sequences that encode proteins and RNA may include introns.

The term “oligonucleotide” typically refers to short polynucleotides,generally, no greater than about 50 nucleotides. It will be understoodthat when a nucleotide sequence is represented by a DNA sequence (i.e.,A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C) inwhich “U” replaces “T.”

By describing two polynucleotides as “operably linked” is meant that asingle-stranded or double-stranded nucleic acid moiety comprises the twopolynucleotides arranged within the nucleic acid moiety in such a mannerthat at least one of the two polynucleotides is able to exert aphysiological effect by which it is characterized upon the other. By wayof example, a promoter operably linked to the coding region of a gene isable to promote transcription of the coding region.

As used herein, “parenteral administration” of a pharmaceuticalcomposition includes any route of administration characterized byphysical breaching of a tissue of a subject and administration of thepharmaceutical composition through the breach in the tissue. Parenteraladministration thus includes, but is not limited to, administration of apharmaceutical composition by injection of the composition, byapplication of the composition through a surgical incision, byapplication of the composition through a tissue-penetrating non-surgicalwound, and the like. In particular, parenteral administration iscontemplated to include, but is not limited to, subcutaneous,intraperitoneal, intramuscular, intrasternal injection, and kidneydialytic infusion techniques.

The term “peptide” typically refers to short polypeptides or to peptidesshorter than the full length native or mature protein.

The term “per application” as used herein refers to administration of adrug or compound to a subject.

The term “pharmaceutical composition” shall mean a compositioncomprising at least one active ingredient, whereby the composition isamenable to investigation for a specified, efficacious outcome in amammal (for example, without limitation, a human). Those of ordinaryskill in the art will understand and appreciate the techniquesappropriate for determining whether an active ingredient has a desiredefficacious outcome based upon the needs of the artisan.

As used herein, the term “pharmaceutically-acceptable carrier” means achemical composition with which an appropriate compound or derivativecan be combined and which, following the combination, can be used toadminister the appropriate compound to a subject.

As used herein, the term “physiologically acceptable” ester or saltmeans an ester or salt form of the active ingredient which is compatiblewith any other ingredients of the pharmaceutical composition, which isnot deleterious to the subject to which the composition is to beadministered.

“Pharmaceutically acceptable” means physiologically tolerable, foreither human or veterinary application.

As used herein, “pharmaceutical compositions” include formulations forhuman and veterinary use.

“Plurality” means at least two.

A “polynucleotide” means a single strand or parallel and anti-parallelstrands of a nucleic acid. Thus, a polynucleotide may be either asingle-stranded or a double-stranded nucleic acid.

“Polypeptide” refers to a polymer composed of amino acid residues,related naturally occurring structural variants, and syntheticnon-naturally occurring analogs thereof linked via peptide bonds,related naturally occurring structural variants, and syntheticnon-naturally occurring analogs thereof.

“Synthetic peptides or polypeptides” means a non-naturally occurringpeptide or polypeptide. Synthetic peptides or polypeptides can besynthesized, for example, using an automated polypeptide synthesizer.Various solid phase peptide synthesis methods are known to those ofskill in the art.

By “presensitization” is meant pre-administration of at least one innateimmune system stimulator prior to challenge with an agent. This issometimes referred to as induction of tolerance.

The term “prevent,” as used herein, means to stop something fromhappening, or taking advance measures against something possible orprobable from happening. In the context of medicine, “prevention”generally refers to action taken to decrease the chance of getting adisease or condition.

A “preventive” or “prophylactic” treatment is a treatment administeredto a subject who does not exhibit signs, or exhibits only early signs,of a disease or disorder. A prophylactic or preventative treatment isadministered for the purpose of decreasing the risk of developingpathology associated with developing the disease or disorder.

“Primer” refers to a polynucleotide that is capable of specificallyhybridizing to a designated polynucleotide template and providing apoint of initiation for synthesis of a complementary polynucleotide.Such synthesis occurs when the polynucleotide primer is placed underconditions in which synthesis is induced, i.e., in the presence ofnucleotides, a complementary polynucleotide template, and an agent forpolymerization such as DNA polymerase. A primer is typicallysingle-stranded, but may be double-stranded. Primers are typicallydeoxyribonucleic acids, but a wide variety of synthetic and naturallyoccurring primers are useful for many applications. A primer iscomplementary to the template to which it is designed to hybridize toserve as a site for the initiation of synthesis, but need not reflectthe exact sequence of the template. In such a case, specifichybridization of the primer to the template depends on the stringency ofthe hybridization conditions. Primers can be labeled with, e.g.,chromogenic, radioactive, or fluorescent moieties and used as detectablemoieties.

As used herein, the term “promoter/regulatory sequence” means a nucleicacid sequence which is required for expression of a gene productoperably linked to the promoter/regulator sequence. In some instances,this sequence may be the core promoter sequence and in other instances,this sequence may also include an enhancer sequence and other regulatoryelements which are required for expression of the gene product. Thepromoter/regulatory sequence may, for example, be one which expressesthe gene product in a tissue specific manner.

A “constitutive” promoter is a promoter which drives expression of agene to which it is operably linked, in a constant manner in a cell. Byway of example, promoters which drive expression of cellularhousekeeping genes are considered to be constitutive promoters.

An “inducible” promoter is a nucleotide sequence which, when operablylinked with a polynucleotide which encodes or specifies a gene product,causes the gene product to be produced in a living cell substantiallyonly when an inducer which corresponds to the promoter is present in thecell.

A “tissue-specific” promoter is a nucleotide sequence which, whenoperably linked with a polynucleotide which encodes or specifies a geneproduct, causes the gene product to be produced in a living cellsubstantially only if the cell is a cell of the tissue typecorresponding to the promoter.

A “prophylactic” treatment is a treatment administered to a subject whodoes not exhibit signs of a disease or exhibits only early signs of thedisease for the purpose of decreasing the risk of developing pathologyassociated with the disease.

As used herein, “protecting group” with respect to a terminal aminogroup refers to a terminal amino group of a peptide, which terminalamino group is coupled with any of various amino-terminal protectinggroups traditionally employed in peptide synthesis. Such protectinggroups include, for example, acyl protecting groups such as formyl,acetyl, benzoyl, trifluoroacetyl, succinyl, and methoxysuccinyl;aromatic urethane protecting groups such as benzyloxycarbonyl; andaliphatic urethane protecting groups, for example, tert-butoxycarbonylor adamantyloxycarbonyl. See Gross and Mienhofer, eds., The Peptides,vol. 3, pp. 3-88 (Academic Press, New York, 1981) for suitableprotecting groups.

As used herein, “protecting group” with respect to a terminal carboxygroup refers to a terminal carboxyl group of a peptide, which terminalcarboxyl group is coupled with any of various carboxyl-terminalprotecting groups. Such protecting groups include, for example,tert-butyl, benzyl or other acceptable groups linked to the terminalcarboxyl group through an ester or ether bond.

The term “protein” typically refers to large polypeptides. Conventionalnotation is used herein to portray polypeptide sequences: the left-handend of a polypeptide sequence is the amino-terminus; the right-hand endof a polypeptide sequence is the carboxyl-terminus.

The term “protein regulatory pathway”, as used herein, refers to boththe upstream regulatory pathway which regulates a protein, as well asthe downstream events which that protein regulates. Such regulationincludes, but is not limited to, transcription, translation, levels,activity, posttranslational modification, and function of the protein ofinterest, as well as the downstream events which the protein regulates.

The terms “protein pathway” and “protein regulatory pathway” are usedinterchangeably herein.

As used herein, the term “purified” and like terms relate to anenrichment of a molecule or compound relative to other componentsnormally associated with the molecule or compound in a nativeenvironment. The term “purified” does not necessarily indicate thatcomplete purity of the particular molecule has been achieved during theprocess. A “highly purified” compound as used herein refers to acompound that is greater than 90% pure. A “significant detectable level”is an amount of contaminate that would be visible in the presented dataand would need to be addressed/explained during analysis of the forensicevidence.

“Recombinant polynucleotide” refers to a polynucleotide having sequencesthat are not naturally joined together. An amplified or assembledrecombinant polynucleotide may be included in a suitable vector, and thevector can be used to transform a suitable host cell.

A recombinant polynucleotide may serve a non-coding function (e.g.,promoter, origin of replication, ribosome-binding site, etc.) as well.

A host cell that comprises a recombinant polynucleotide is referred toas a “recombinant host cell.” A gene which is expressed in a recombinanthost cell wherein the gene comprises a recombinant polynucleotide,produces a “recombinant polypeptide.”

A “recombinant polypeptide” is one which is produced upon expression ofa recombinant polynucleotide.

A “receptor” is a compound that specifically binds to a ligand.

A “ligand” is a compound that specifically binds to a target receptor.

A “recombinant cell” is a cell that comprises a transgene. Such a cellmay be a eukaryotic or a prokaryotic cell. Also, the transgenic cellencompasses, but is not limited to, an embryonic stem cell comprisingthe transgene, a cell obtained from a chimeric mammal derived from atransgenic embryonic stem cell where the cell comprises the transgene, acell obtained from a transgenic mammal, or fetal or placental tissuethereof, and a prokaryotic cell comprising the transgene.

The term “regulate” refers to either stimulating or inhibiting afunction or activity of interest.

As used herein, the term “reporter gene” means a gene, the expression ofwhich can be detected using a known method. By way of example, theEscherichia coli lacZ gene may be used as a reporter gene in a mediumbecause expression of the lacZ gene can be detected using known methodsby adding the chromogenic substrate o-nitrophenyl-β-galactoside to themedium (Gerhardt et al., eds., 1994, Methods for General and MolecularBacteriology, American Society for Microbiology, Washington, D.C., p.574).

A “sample,” as used herein, refers preferably to a biological samplefrom a subject for which an assay or other use is needed, including, butnot limited to, normal tissue samples, diseased tissue samples, sputum,mucus, phlegm, biopsies, cerebrospinal fluid, blood, serum, plasma,other blood components, gastric aspirates, throat swabs, pleuraleffusion, peritoneal fluid, follicular fluid, ascites, skin, hair,tissue, blood, plasma, cells, saliva, sweat, tears, semen, stools, Papsmears, and urine. A sample can also be any other source of materialobtained from a subject which contains cells, tissues, or fluid ofinterest. A sample can also be obtained from cell or tissue culture.

As used herein, the term “secondary antibody” refers to an antibody thatbinds to the constant region of another antibody (the primary antibody).

By the term “signal sequence” is meant a polynucleotide sequence whichencodes a peptide that directs the path a polypeptide takes within acell, i.e., it directs the cellular processing of a polypeptide in acell, including, but not limited to, eventual secretion of a polypeptidefrom a cell. A signal sequence is a sequence of amino acids which aretypically, but not exclusively, found at the amino terminus of apolypeptide which targets the synthesis of the polypeptide to theendoplasmic reticulum. In some instances, the signal peptide isproteolytically removed from the polypeptide and is thus absent from themature protein.

By “small interfering RNAs (siRNAs)” is meant, inter alia, an isolateddsRNA molecule comprised of both a sense and an anti-sense strand. Inone aspect, it is greater than 10 nucleotides in length. siRNA alsorefers to a single transcript which has both the sense and complementaryantisense sequences from the target gene, e.g., a hairpin. siRNA furtherincludes any form of dsRNA (proteolytically cleaved products of largerdsRNA, partially purified RNA, essentially pure RNA, synthetic RNA,recombinantly produced RNA) as well as altered RNA that differs fromnaturally occurring RNA by the addition, deletion, substitution, and/oralteration of one or more nucleotides.

As used herein, the term “solid support” relates to a solvent insolublesubstrate that is capable of forming linkages (preferably covalentbonds) with various compounds. The support can be either biological innature, such as, without limitation, a cell or bacteriophage particle,or synthetic, such as, without limitation, an acrylamide derivative,agarose, cellulose, nylon, silica, or magnetized particles.

By the term “specifically binds to”, as used herein, is meant when acompound or ligand functions in a binding reaction or assay conditionswhich is determinative of the presence of the compound in a sample ofheterogeneous compounds.

The term “standard,” as used herein, refers to something used forcomparison. For example, it can be a known standard agent or compoundwhich is administered and used for comparing results when administeringa test compound, or it can be a standard parameter or function which ismeasured to obtain a control value when measuring an effect of an agentor compound on a parameter or function. Standard can also refer to an“internal standard”, such as an agent or compound which is added atknown amounts to a sample and is useful in determining such things aspurification or recovery rates when a sample is processed or subjectedto purification or extraction procedures before a marker of interest ismeasured. Internal standards are often a purified marker of interestwhich has been labeled, such as with a radioactive isotope, allowing itto be distinguished from an endogenous marker.

A “subject” of analysis, diagnosis, or treatment is an animal. Suchanimals include mammals, preferably a human.

As used herein, a “subject in need thereof” is a patient, animal,mammal, or human, who will benefit from the method of this invention.

As used herein, a “substantially homologous amino acid sequences”includes those amino acid sequences which have at least about 95%homology, preferably at least about 96% homology, more preferably atleast about 97% homology, even more preferably at least about 98%homology, and most preferably at least about 99% or more homology to anamino acid sequence of a reference antibody chain. Amino acid sequencesimilarity or identity can be computed by using the BLASTP and TBLASTNprograms which employ the BLAST (basic local alignment search tool)2.0.14 algorithm. The default settings used for these programs aresuitable for identifying substantially similar amino acid sequences forpurposes of the present invention.

“Substantially homologous nucleic acid sequence” means a nucleic acidsequence corresponding to a reference nucleic acid sequence wherein thecorresponding sequence encodes a peptide having substantially the samestructure and function as the peptide encoded by the reference nucleicacid sequence; e.g., where only changes in amino acids not significantlyaffecting the peptide function occur. Preferably, the substantiallyidentical nucleic acid sequence encodes the peptide encoded by thereference nucleic acid sequence. The percentage of identity between thesubstantially similar nucleic acid sequence and the reference nucleicacid sequence is at least about 50%, 65%, 75%, 85%, 95%, 99% or more.Substantial identity of nucleic acid sequences can be determined bycomparing the sequence identity of two sequences, for example byphysical/chemical methods (i.e., hybridization) or by sequence alignmentvia computer algorithm. Suitable nucleic acid hybridization conditionsto determine if a nucleotide sequence is substantially similar to areference nucleotide sequence are: 7% sodium dodecyl sulfate SDS, 0.5 MNaPO₄, 1 mM EDTA at 50° C. with washing in 2× standard saline citrate(SSC), 0.1% SDS at 50° C.; preferably in 7% (SDS), 0.5 M NaPO₄, 1 mMEDTA at 50° C. with washing in 1×SSC, 0.1% SDS at 50° C.; preferably 7%SDS, 0.5 M NaPO₄, 1 mM EDTA at 50° C. with washing in 0.5×SSC, 0.1% SDSat 50° C.; and more preferably in 7% SDS, 0.5 M NaPO₄, 1 mM EDTA at 50°C. with washing in 0.1×SSC, 0.1% SDS at 65° C. Suitable computeralgorithms to determine substantial similarity between two nucleic acidsequences include, GCS program package (Devereux et al., 1984 Nucl.Acids Res. 12:387), and the BLASTN or FASTA programs (Altschul et al.,1990 Proc. Natl. Acad. Sci. USA. 1990 87:14:5509-13; Altschul et al., J.Mol. Biol. 1990 215:3:403-10; Altschul et al., 1997 Nucleic Acids Res.25:3389-3402). The default settings provided with these programs aresuitable for determining substantial similarity of nucleic acidsequences for purposes of the present invention.

The term “substantially pure” describes a compound, e.g., a protein orpolypeptide which has been separated from components which naturallyaccompany it. Typically, a compound is substantially pure when at least10%, more preferably at least 20%, more preferably at least 50%, morepreferably at least 60%, more preferably at least 75%, more preferablyat least 90%, and most preferably at least 99% of the total material (byvolume, by wet or dry weight, or by mole percent or mole fraction) in asample is the compound of interest. Purity can be measured by anyappropriate method, e.g., in the case of polypeptides by columnchromatography, gel electrophoresis, or HPLC analysis. A compound, e.g.,a protein, is also substantially purified when it is essentially free ofnaturally associated components or when it is separated from the nativecontaminants which accompany it in its natural state.

The term “symptom,” as used herein, refers to any morbid phenomenon ordeparture from the normal in structure, function, or sensation,experienced by the patient and indicative of disease. In contrast, a“sign” is objective evidence of disease. For example, a bloody nose is asign. It is evident to the patient, doctor, nurse and other observers.

A “therapeutic” treatment is a treatment administered to a subject whoexhibits signs of pathology for the purpose of diminishing oreliminating those signs.

A “therapeutically effective amount” of a compound is that amount ofcompound which is sufficient to provide a beneficial effect to thesubject to which the compound is administered.

As used herein, the term “transgene” means an exogenous nucleic acidsequence comprising a nucleic acid which encodes a promoter/regulatorysequence operably linked to nucleic acid which encodes an amino acidsequence, which exogenous nucleic acid is encoded by a transgenicmammal.

As used herein, the term “transgenic mammal” means a mammal, the germcells of which comprise an exogenous nucleic acid.

As used herein, a “transgenic cell” is any cell that comprises a nucleicacid sequence that has been introduced into the cell in a manner thatallows expression of a gene encoded by the introduced nucleic acidsequence.

The term to “treat,” as used herein, means reducing the frequency withwhich symptoms are experienced by a patient or subject or administeringan agent or compound to reduce the frequency with which symptoms areexperienced.

A “prophylactic” treatment is a treatment administered to a subject whodoes not exhibit signs of a disease or exhibits only early signs of thedisease for the purpose of decreasing the risk of developing pathologyassociated with the disease.

A “variant”, as described herein, refers to a segment of DNA thatdiffers from the reference DNA. A “marker” or a “polymorphic marker”, asdefined herein, is a variant. Alleles that differ from the reference arereferred to as “variant” alleles.

A “vector” is a composition of matter which comprises an isolatednucleic acid and which can be used to deliver the isolated nucleic acidto the interior of a cell. Numerous vectors are known in the artincluding, but not limited to, linear polynucleotides, polynucleotidesassociated with ionic or amphiphilic compounds, plasmids, and viruses.Thus, the term “vector” includes an autonomously replicating plasmid ora virus. The term should also be construed to include non-plasmid andnon-viral compounds which facilitate transfer or delivery of nucleicacid to cells, such as, for example, polylysine compounds, liposomes,and the like. Examples of viral vectors include, but are not limited to,adenoviral vectors, adeno-associated virus vectors, retroviral vectors,recombinant viral vectors, and the like. Examples of non-viral vectorsinclude, but are not limited to, liposomes, polyamine derivatives of DNAand the like.

“Expression vector” refers to a vector comprising a recombinantpolynucleotide comprising expression control sequences operativelylinked to a nucleotide sequence to be expressed. An expression vectorcomprises sufficient cis-acting elements for expression; other elementsfor expression can be supplied by the host cell or in an in vitroexpression system. Expression vectors include all those known in theart, such as cosmids, plasmids (e.g., naked or contained in liposomes)and viruses that incorporate the recombinant polynucleotide.

Embodiments

In one embodiment, a hepcidin can be used for treatment of inflammatoryconditions. In one aspect, it is useful for preventing or treating renalischemia reperfusion injury. In one aspect, it is useful for preventingor treating acute kidney injury.

One of ordinary skill in the art will appreciate that the sequences ofthe components of the hepcidin protein can be modified independently ofone another with conservative amino acid changes, including, insertions,deletions, and substitutions, and that the valency could be altered aswell, as long as the resulting multimer/multimeric complex remainseffective. Amino acid changes (fragments and homologs) can be madeindependently in each hepcidin as well when they are being used incombination therapy.

In one aspect, a protein or peptide of the invention, or a combinationthereof, can be administered by a route selected from, including, butnot limited to, intravenously, intrathecally, locally, intramuscularly,topically, orally, intra-arterially, parenterally, etc. Administrationcan be more than once. One of ordinary skill in the art can determinehow often to administer the compound, the dose to be used, and whatcombination of other agents it can be administered with such astherapeutic agents and/or other drugs or compounds such as antimicrobialagents, anti-inflammatory agents, etc. One of ordinary skill in the willbe able to determine when or if to use an additional agent and the routeof administration.

The peptides of the present invention may be readily prepared bystandard, well-established techniques, such as solid-phase peptidesynthesis (SPPS) as described by Stewart et al. in Solid Phase PeptideSynthesis, 2nd Edition, 1984, Pierce Chemical Company, Rockford, Ill.;and as described by Bodanszky and Bodanszky in The Practice of PeptideSynthesis, 1984, Springer-Verlag, New York. At the outset, a suitablyprotected amino acid residue is attached through its carboxyl group to aderivatized, insoluble polymeric support, such as cross-linkedpolystyrene or polyamide resin. “Suitably protected” refers to thepresence of protecting groups on both the α-amino group of the aminoacid, and on any side chain functional groups. Side chain protectinggroups are generally stable to the solvents, reagents and reactionconditions used throughout the synthesis, and are removable underconditions that will not affect the final peptide product. Stepwisesynthesis of the oligopeptide is carried out by the removal of theN-protecting group from the initial amino acid, and couple thereto ofthe carboxyl end of the next amino acid in the sequence of the desiredpeptide. This amino acid is also suitably protected. The carboxyl of theincoming amino acid can be activated to react with the N-terminus of thesupport-bound amino acid by formation into a reactive group such asformation into a carbodiimide, a symmetric acid anhydride or an “activeester” group such as hydroxybenzotriazole or pentafluorophenly esters.

Examples of solid phase peptide synthesis methods include the BOC methodthat utilized tert-butyloxcarbonyl as the α-amino protecting group, andthe FMOC method which utilizes 9-fluorenylmethyloxycarbonyl to protectthe α-amino of the amino acid residues, both methods of which arewell-known by those of skill in the art.

To ensure that the proteins or peptides obtained from either chemical orbiological synthetic techniques is the desired peptide, analysis of thepeptide composition should be conducted. Such amino acid compositionanalysis may be conducted using high resolution mass spectrometry todetermine the molecular weight of the peptide. Alternatively, oradditionally, the amino acid content of the peptide can be confirmed byhydrolyzing the peptide in aqueous acid, and separating, identifying andquantifying the components of the mixture using HPLC, or an amino acidanalyzer. Protein sequenators, which sequentially degrade the peptideand identify the amino acids in order, may also be used to determinedefinitely the sequence of the peptide.

Prior to its use, the peptide can be purified to remove contaminants. Inthis regard, it will be appreciated that the peptide will be purified tomeet the standards set out by the appropriate regulatory agencies. Anyone of a number of a conventional purification procedures may be used toattain the required level of purity including, for example,reversed-phase high-pressure liquid chromatography (HPLC) using analkylated silica column such as C₄-, C₈- or C₁₈-silica. A gradientmobile phase of increasing organic content is generally used to achievepurification, for example, acetonitrile in an aqueous buffer, usuallycontaining a small amount of trifluoroacetic acid. Ion-exchangechromatography can be also used to separate peptides based on theircharge.

Substantially pure peptide obtained as described herein may be purifiedby following known procedures for protein purification, wherein animmunological, enzymatic or other assay is used to monitor purificationat each stage in the procedure. Protein purification methods are wellknown in the art, and are described, for example in Deutscher et al.(ed., 1990, Guide to Protein Purification, Harcourt Brace Jovanovich,San Diego).

Peptide Modification and Preparation

Peptide preparation is described in the Examples. It will beappreciated, of course, that the proteins or peptides of the inventionmay incorporate amino acid residues which are modified without affectingactivity. For example, the termini may be derivatized to includeblocking groups, i.e. chemical substituents suitable to protect and/orstabilize the N- and C-termini from “undesirable degradation”, a termmeant to encompass any type of enzymatic, chemical or biochemicalbreakdown of the compound at its termini which is likely to affect thefunction of the compound, i.e. sequential degradation of the compound ata terminal end thereof.

Blocking groups include protecting groups conventionally used in the artof peptide chemistry which will not adversely affect the in vivoactivities of the peptide. For example, suitable N-terminal blockinggroups can be introduced by alkylation or acylation of the N-terminus.Examples of suitable N-terminal blocking groups include C₁-C₅ branchedor unbranched alkyl groups, acyl groups such as formyl and acetylgroups, as well as substituted forms thereof, such as theacetamidomethyl (Acm) group. Desamino analogs of amino acids are alsouseful N-terminal blocking groups, and can either be coupled to theN-terminus of the peptide or used in place of the N-terminal reside.Suitable C-terminal blocking groups, in which the carboxyl group of theC-terminus is either incorporated or not, include esters, ketones oramides. Ester or ketone-forming alkyl groups, particularly lower alkylgroups such as methyl, ethyl and propyl, and amide-forming amino groupssuch as primary amines (—NH₂), and mono- and di-alkylamino groups suchas methylamino, ethylamino, dimethylamino, diethylamino,methylethylamino and the like are examples of C-terminal blockinggroups. Descarboxylated amino acid analogues such as agmatine are alsouseful C-terminal blocking groups and can be either coupled to thepeptide's C-terminal residue or used in place of it. Further, it will beappreciated that the free amino and carboxyl groups at the termini canbe removed altogether from the peptide to yield desamino anddescarboxylated forms thereof without affect on peptide activity.

Acid addition salts of the present invention are also contemplated asfunctional equivalents. Thus, a peptide in accordance with the presentinvention treated with an inorganic acid such as hydrochloric,hydrobromic, sulfuric, nitric, phosphoric, and the like, or an organicacid such as an acetic, propionic, glycolic, pyruvic, oxalic, malic,malonic, succinic, maleic, fumaric, tataric, citric, benzoic, cinnamie,mandelic, methanesulfonic, ethanesulfonic, p-toluenesulfonic, salicyclicand the like, to provide a water soluble salt of the peptide is suitablefor use in the invention.

The present invention also provides for analogs of proteins. Analogs candiffer from naturally occurring proteins or peptides by conservativeamino acid sequence differences or by modifications which do not affectsequence, or by both.

For example, conservative amino acid changes may be made, which althoughthey alter the primary sequence of the protein or peptide, do notnormally alter its function. To that end, 10 or more conservative aminoacid changes typically have no effect on peptide function.

Modifications (which do not normally alter primary sequence) include invivo, or in vitro chemical derivatization of polypeptides, e.g.,acetylation, or carboxylation. Also included are modifications ofglycosylation, e.g., those made by modifying the glycosylation patternsof a polypeptide during its synthesis and processing or in furtherprocessing steps; e.g., by exposing the polypeptide to enzymes whichaffect glycosylation, e.g., mammalian glycosylating or deglycosylatingenzymes. Also embraced are sequences which have phosphorylated aminoacid residues, e.g., phosphotyrosine, phosphoserine, orphosphothreonine.

Also included are polypeptides which have been modified using ordinarymolecular biological techniques so as to improve their resistance toproteolytic degradation or to optimize solubility properties or torender them more suitable as a therapeutic agent. Analogs of suchpolypeptides include those containing residues other than naturallyoccurring L-amino acids, e.g., D-amino acids or non-naturally occurringor non-standard synthetic amino acids. The peptides of the invention arenot limited to products of any of the specific exemplary processeslisted herein.

The invention includes the use of beta-alanine (also referred to asβ-alanine, β-Ala, bA, and βA, having the structure:

Sequences are provided herein which use the symbol “βA”, but in theSequence Listing submitted herewith “βA” is provided as “Xaa” andreference in the text of the Sequence Listing indicates that Xaa is betaalanine.

Peptides useful in the present invention, such as standards, ormodifications for analysis, may be readily prepared by standard,well-established techniques, such as solid-phase peptide synthesis(SPPS) as described by Stewart et al. in Solid Phase Peptide Synthesis,2nd Edition, 1984, Pierce Chemical Company, Rockford, Ill.; and asdescribed by Bodanszky and Bodanszky in The Practice of PeptideSynthesis, 1984, Springer-Verlag, New York. At the outset, a suitablyprotected amino acid residue is attached through its carboxyl group to aderivatized, insoluble polymeric support, such as cross-linkedpolystyrene or polyamide resin. “Suitably protected” refers to thepresence of protecting groups on both the α-amino group of the aminoacid, and on any side chain functional groups. Side chain protectinggroups are generally stable to the solvents, reagents and reactionconditions used throughout the synthesis, and are removable underconditions which will not affect the final peptide product. Stepwisesynthesis of the oligopeptide is carried out by the removal of theN-protecting group from the initial amino acid, and couple thereto ofthe carboxyl end of the next amino acid in the sequence of the desiredpeptide. This amino acid is also suitably protected. The carboxyl of theincoming amino acid can be activated to react with the N-terminus of thesupport-bound amino acid by formation into a reactive group such asformation into a carbodiimide, a symmetric acid anhydride or an “activeester” group such as hydroxybenzotriazole or pentafluorophenly esters.

Examples of solid phase peptide synthesis methods include the BOC methodwhich utilized tert-butyloxcarbonyl as the α-amino protecting group, andthe FMOC method which utilizes 9-fluorenylmethyloxycarbonyl to protectthe α-amino of the amino acid residues, both methods of which arewell-known by those of skill in the art.

Incorporation of N- and/or C-blocking groups can also be achieved usingprotocols conventional to solid phase peptide synthesis methods. Forincorporation of C-terminal blocking groups, for example, synthesis ofthe desired peptide is typically performed using, as solid phase, asupporting resin that has been chemically modified so that cleavage fromthe resin results in a peptide having the desired C-terminal blockinggroup. To provide peptides in which the C-terminus bears a primary aminoblocking group, for instance, synthesis is performed using ap-methylbenzhydrylamine (MBHA) resin so that, when peptide synthesis iscompleted, treatment with hydrofluoric acid releases the desiredC-terminally amidated peptide. Similarly, incorporation of anN-methylamine blocking group at the C-terminus is achieved usingN-methylaminoethyl-derivatized DVB, resin, which upon HF treatmentreleases a peptide bearing an N-methylamidated C-terminus. Blockage ofthe C-terminus by esterification can also be achieved using conventionalprocedures. This entails use of resin/blocking group combination thatpermits release of side-chain peptide from the resin, to allow forsubsequent reaction with the desired alcohol, to form the esterfunction. FMOC protecting group, in combination with DVB resinderivatized with methoxyalkoxybenzyl alcohol or equivalent linker, canbe used for this purpose, with cleavage from the support being effectedby TFA in dichloromethane. Esterification of the suitably activatedcarboxyl function e.g. with DCC, can then proceed by addition of thedesired alcohol, followed by deprotection and isolation of theesterified peptide product.

Incorporation of N-terminal blocking groups can be achieved while thesynthesized peptide is still attached to the resin, for instance bytreatment with a suitable anhydride and nitrile. To incorporate anacetyl blocking group at the N-terminus, for instance, the resin-coupledpeptide can be treated with 20% acetic anhydride in acetonitrile. TheN-blocked peptide product can then be cleaved from the resin,deprotected and subsequently isolated.

To ensure that the peptide obtained from either chemical or biologicalsynthetic techniques is the desired peptide, analysis of the peptidecomposition should be conducted. Such amino acid composition analysismay be conducted using high resolution mass spectrometry to determinethe molecular weight of the peptide. Alternatively, or additionally, theamino acid content of the peptide can be confirmed by hydrolyzing thepeptide in aqueous acid, and separating, identifying and quantifying thecomponents of the mixture using HPLC, or an amino acid analyzer. Proteinsequenators, which sequentially degrade the peptide and identify theamino acids in order, may also be used to determine definitely thesequence of the peptide.

Prior to its use, the peptide may be purified to remove contaminants. Inthis regard, it will be appreciated that the peptide will be purified soas to meet the standards set out by the appropriate regulatory agencies.Any one of a number of a conventional purification procedures may beused to attain the required level of purity including, for example,reversed-phase high performance liquid chromatography (HPLC) using analkylated silica column such as C₄-, C₈- or C₁₈-silica. A gradientmobile phase of increasing organic content is generally used to achievepurification, for example, acetonitrile in an aqueous buffer, usuallycontaining a small amount of trifluoroacetic acid. Ion-exchangechromatography can be also used to separate peptides based on theircharge.

It will be appreciated, of course, that the peptides or antibodies,derivatives, or fragments thereof may incorporate amino acid residueswhich are modified without affecting activity. For example, the terminimay be derivatized to include blocking groups, i.e. chemicalsubstituents suitable to protect and/or stabilize the N- and C-terminifrom “undesirable degradation”, a term meant to encompass any type ofenzymatic, chemical or biochemical breakdown of the compound at itstermini which is likely to affect the function of the compound, i.e.sequential degradation of the compound at a terminal end thereof.

Blocking groups include protecting groups conventionally used in the artof peptide chemistry which will not adversely affect the in vivoactivities of the peptide.

For example, suitable N-terminal blocking groups can be introduced byalkylation or acylation of the N-terminus. Examples of suitableN-terminal blocking groups include C₁-C₅ branched or unbranched alkylgroups, acyl groups such as formyl and acetyl groups, as well assubstituted forms thereof, such as the acetamidomethyl (Acm) group.Desamino analogs of amino acids are also useful N-terminal blockinggroups, and can either be coupled to the N-terminus of the peptide orused in place of the N-terminal reside. Suitable C-terminal blockinggroups, in which the carboxyl group of the C-terminus is eitherincorporated or not, include esters, ketones or amides. Ester orketone-forming alkyl groups, particularly lower alkyl groups such asmethyl, ethyl and propyl, and amide-forming amino groups such as primaryamines (—NH₂), and mono- and di-alkylamino groups such as methylamino,ethylamino, dimethylamino, diethylamino, methylethylamino and the likeare examples of C-terminal blocking groups. Descarboxylated amino acidanalogues such as agmatine are also useful C-terminal blocking groupsand can be either coupled to the peptide's C-terminal residue or used inplace of it. Further, it will be appreciated that the free amino andcarboxyl groups at the termini can be removed altogether from thepeptide to yield desamino and descarboxylated forms thereof withoutaffect on peptide activity.

Other modifications can also be incorporated without adversely affectingthe activity and these include, but are not limited to, substitution ofone or more of the amino acids in the natural L-isomeric form with aminoacids in the D-isomeric form. Thus, the peptide may include one or moreD-amino acid resides, or may comprise amino acids which are all in theD-form. Retro-inverso forms of peptides in accordance with the presentinvention are also contemplated, for example, inverted peptides in whichall amino acids are substituted with D-amino acid forms.

Substantially pure protein obtained as described herein may be purifiedby following known procedures for protein purification, wherein animmunological, enzymatic or other assay is used to monitor purificationat each stage in the procedure. Protein purification methods are wellknown in the art, and are described, for example in Deutscher et al.(ed., 1990, Guide to Protein Purification, Harcourt Brace Jovanovich,San Diego).

As discussed, modifications or optimizations of peptide ligands of theinvention are within the scope of the application. Modified or optimizedpeptides are included within the definition of peptide binding ligand.Specifically, a peptide sequence identified can be modified to optimizeits potency, pharmacokinetic behavior, stability and/or otherbiological, physical and chemical properties.

Amino Acid Substitutions

In certain embodiments, the disclosed methods and compositions mayinvolve preparing peptides with one or more substituted amino acidresidues.

In various embodiments, the structural, physical and/or therapeuticcharacteristics of peptide sequences may be optimized by replacing oneor more amino acid residues.

Other modifications can also be incorporated without adversely affectingthe activity and these include, but are not limited to, substitution ofone or more of the amino acids in the natural L-isomeric form with aminoacids in the D-isomeric form. Thus, the peptide may include one or moreD-amino acid resides, or may comprise amino acids which are all in theD-form. Retro-inverso forms of peptides in accordance with the presentinvention are also contemplated, for example, inverted peptides in whichall amino acids are substituted with D-amino acid forms.

The skilled artisan will be aware that, in general, amino acidsubstitutions in a peptide typically involve the replacement of an aminoacid with another amino acid of relatively similar properties (i.e.,conservative amino acid substitutions). The properties of the variousamino acids and effect of amino acid substitution on protein structureand function have been the subject of extensive study and knowledge inthe art. For example, one can make the following isosteric and/orconservative amino acid changes in the parent polypeptide sequence withthe expectation that the resulting polypeptides would have a similar orimproved profile of the properties described above:

Substitution of alkyl-substituted hydrophobic amino acids: includingalanine, leucine, isoleucine, valine, norleucine, S-2-aminobutyric acid,S-cyclohexylalanine or other simple alpha-amino acids substituted by analiphatic side chain from C1-10 carbons including branched, cyclic andstraight chain alkyl, alkenyl or alkynyl substitutions.

Substitution of aromatic-substituted hydrophobic amino acids: includingphenylalanine, tryptophan, tyrosine, biphenylalanine, 1-naphthylalanine,2-naphthylalanine, 2-benzothienylalanine, 3-benzothienylalanine,histidine, amino, alkylamino, dialkylamino, aza, halogenated (fluoro,chloro, bromo, or iodo) or alkoxy-substituted forms of the previouslisted aromatic amino acids, illustrative examples of which are: 2-, 3-or 4-aminophenylalanine, 2-, 3- or 4-chlorophenylalanine, 2-, 3- or4-methylphenylalanine, 2-, 3- or 4-methoxyphenylalanine, 5-amino-,5-chloro-, 5-methyl- or 5-methoxytryptophan, 2′-, 3′-, or 4′-amino-,2′-, 3′-, or 4′-chloro-, 2, 3, or 4-biphenylalanine, 2′-, 3′-, or4′-methyl-2, 3 or 4-biphenylalanine, and 2- or 3-pyridylalanine.

Substitution of amino acids containing basic functions: includingarginine, lysine, histidine, ornithine, 2,3-diaminopropionic acid,homoarginine, alkyl, alkenyl, or aryl-substituted (from C₁-C₁₀ branched,linear, or cyclic) derivatives of the previous amino acids, whether thesubstituent is on the heteroatoms (such as the alpha nitrogen, or thedistal nitrogen or nitrogens, or on the alpha carbon, in the pro-Rposition for example. Compounds that serve as illustrative examplesinclude: N-epsilon-isopropyl-lysine, 3-(4-tetrahydropyridyl)-glycine,3-(4-tetrahydropyridyl)-alanine, N,N-gamma, gamma′-diethyl-homoarginine.Included also are compounds such as alpha methyl arginine, alpha methyl2,3-diaminopropionic acid, alpha methyl histidine, alpha methylornithine where alkyl group occupies the pro-R position of the alphacarbon. Also included are the amides formed from alkyl, aromatic,heteroaromatic (where the heteroaromatic group has one or morenitrogens, oxygens, or sulfur atoms singly or in combination) carboxylicacids or any of the many well-known activated derivatives such as acidchlorides, active esters, active azolides and related derivatives) andlysine, ornithine, or 2,3-diaminopropionic acid.

Substitution of acidic amino acids: including aspartic acid, glutamicacid, homoglutamic acid, tyrosine, alkyl, aryl, arylalkyl, andheteroaryl sulfonamides of 2,4-diaminopriopionic acid, ornithine orlysine and tetrazole-substituted alkyl amino acids.

Substitution of side chain amide residues: including asparagine,glutamine, and alkyl or aromatic substituted derivatives of asparagineor glutamine.

Substitution of hydroxyl containing amino acids: including serine,threonine, homoserine, 2,3-diaminopropionic acid, and alkyl or aromaticsubstituted derivatives of serine or threonine. It is also understoodthat the amino acids within each of the categories listed above can besubstituted for another of the same group.

For example, the hydropathic index of amino acids may be considered(Kyte & Doolittle, 1982, J. Mol. Biol., 157:105-132). The relativehydropathic character of the amino acid contributes to the secondarystructure of the resultant protein, which in turn defines theinteraction of the protein with other molecules. Each amino acid hasbeen assigned a hydropathic index on the basis of its hydrophobicity andcharge characteristics (Kyte & Doolittle, 1982), these are: isoleucine(+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8);cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine(−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine(−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine(−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine(−4.5). In making conservative substitutions, the use of amino acidswhose hydropathic indices are within +/−2 is preferred, within +/−1 aremore preferred, and within +/−0.5 are even more preferred.

Amino acid substitution may also take into account the hydrophilicity ofthe amino acid residue (e.g., U.S. Pat. No. 4,554,101). Hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0); glutamate (+3.0); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5.+−0.1); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4). Replacement ofamino acids with others of similar hydrophilicity is preferred.

Other considerations include the size of the amino acid side chain. Forexample, it would generally not be preferred to replace an amino acidwith a compact side chain, such as glycine or serine, with an amino acidwith a bulky side chain, e.g., tryptophan or tyrosine. The effect ofvarious amino acid residues on protein secondary structure is also aconsideration. Through empirical study, the effect of different aminoacid residues on the tendency of protein domains to adopt analpha-helical, beta-sheet or reverse turn secondary structure has beendetermined and is known in the art (see, e.g., Chou & Fasman, 1974,Biochemistry, 13:222-245; 1978, Ann. Rev. Biochem., 47: 251-276; 1979,Biophys. J., 26:367-384).

Based on such considerations and extensive empirical study, tables ofconservative amino acid substitutions have been constructed and areknown in the art. For example: arginine and lysine; glutamate andaspartate; serine and threonine; glutamine and asparagine; and valine,leucine and isoleucine. Alternatively: Ala (A) leu, ile, val; Arg (R)gln, asn, lys; Asn (N) his, asp, lys, arg, gln; Asp (D) asn, glu; Cys(C) ala, ser; Gln (Q) glu, asn; Glu (E) gln, asp; Gly (G) ala; His (H)asn, gln, lys, arg; Ile (I) val, met, ala, phe, leu; Leu (L) val, met,ala, phe, ile; Lys (K) gln, asn, arg; Met (M) phe, ile, leu; Phe (F)leu, val, ile, ala, tyr; Pro (P) ala; Ser (S), thr; Thr (T) ser; Trp (W)phe, tyr; Tyr (Y) trp, phe, thr, ser; Val (V) ile, leu, met, phe, ala.

Other considerations for amino acid substitutions include whether or notthe residue is located in the interior of a protein or is solventexposed. For interior residues, conservative substitutions wouldinclude: Asp and Asn; Ser and Thr; Ser and Ala; Thr and Ala; Ala andGly; Ile and Val; Val and Leu; Leu and Ile; Leu and Met; Phe and Tyr;Tyr and Trp. (See, e.g., PROWL Rockefeller University website). Forsolvent exposed residues, conservative substitutions would include: Aspand Asn; Asp and Glu; Glu and Gln; Glu and Ala; Gly and Asn; Ala andPro; Ala and Gly; Ala and Ser; Ala and Lys; Ser and Thr; Lys and Arg;Val and Leu; Leu and Ile; Ile and Val; Phe and Tyr. Various matriceshave been constructed to assist in selection of amino acidsubstitutions, such as the PAM250 scoring matrix, Dayhoff matrix,Grantham matrix, McLachlan matrix, Doolittle matrix, Henikoff matrix,Miyata matrix, Fitch matrix, Jones matrix, Rao matrix, Levin matrix andRisler matrix (Idem.)

In determining amino acid substitutions, one may also consider theexistence of intermolecular or intramolecular bonds, such as formationof ionic bonds (salt bridges) between positively charged residues (e.g.,His, Arg, Lys) and negatively charged residues (e.g., Asp, Glu) ordisulfide bonds between nearby cysteine residues.

Methods of substituting any amino acid for any other amino acid in anencoded peptide sequence are well known and a matter of routineexperimentation for the skilled artisan, for example by the technique ofsite-directed mutagenesis or by synthesis and assembly ofoligonucleotides encoding an amino acid substitution and splicing intoan expression vector construct.

Pharmaceutical Compositions and Administration

The invention is also directed to methods of administering the compoundsof the invention to a subject.

Pharmaceutical compositions comprising the present compounds areadministered to a subject in need thereof by any number of routesincluding, but not limited to, topical, oral, intravenous,intramuscular, intra-arterial, intramedullary, intrathecal,intraventricular, transdermal, subcutaneous, intraperitoneal,intranasal, enteral, topical, sublingual, or rectal means.

In accordance with one embodiment, a method of treating a subject inneed of such treatment is provided. The method comprises administering apharmaceutical composition comprising at least one compound of thepresent invention to a subject in need thereof. Compounds identified bythe methods of the invention can be administered with known compounds orother medications as well.

The pharmaceutical compositions useful for practicing the invention maybe administered to deliver a dose of between 1 ng/kg/day and 100mg/kg/day.

The invention encompasses the preparation and use of pharmaceuticalcompositions comprising a compound useful for treatment of the diseasesand disorders disclosed herein as an active ingredient. Such apharmaceutical composition may consist of the active ingredient alone,in a form suitable for administration to a subject, or thepharmaceutical composition may comprise the active ingredient and one ormore pharmaceutically acceptable carriers, one or more additionalingredients, or some combination of these. The active ingredient may bepresent in the pharmaceutical composition in the form of aphysiologically acceptable ester or salt, such as in combination with aphysiologically acceptable cation or anion, as is well known in the art.

As used herein, the term “physiologically acceptable” ester or saltmeans an ester or salt form of the active ingredient which is compatiblewith any other ingredients of the pharmaceutical composition, which isnot deleterious to the subject to which the composition is to beadministered.

The compositions of the present invention may comprise at least oneactive peptide, one or more acceptable carriers, and optionally otherpeptides or therapeutic agents.

For in vivo applications, the peptides of the present invention maycomprise a pharmaceutically acceptable salt. Suitable acids which arecapable of forming such salts with the compounds of the presentinvention include inorganic acids such as hydrochloric acid, hydrobromicacid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid,phosphoric acid and the like; and organic acids such as formic acid,acetic acid, propionic acid, glycolic acid, lactic acid, anthranilicacid, cinnamic acid, naphthalene sulfonic acid, sulfanilic acid and thelike.

Pharmaceutically acceptable carriers include physiologically tolerableor acceptable diluents, excipients, solvents or adjuvants. Thecompositions are preferably sterile and nonpyrogenic. Examples ofsuitable carriers include, but are not limited to, water, normal saline,dextrose, mannitol, lactose or other sugars, lecithin, albumin, sodiumglutamate, cysteine hydrochloride, ethanol, polyols (propylene glycol,polyethylene glycol, glycerol, and the like), vegetable oils (such asolive oil), injectable organic esters such as ethyl oleate, ethoxylatedisostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters,microcrystalline cellulose, aluminum metahydroxide, bentonite, kaolin,agar-agar and tragacanth, or mixtures of these substances, and the like.

The pharmaceutical compositions may also contain minor amounts ofnontoxic auxiliary pharmaceutical substances or excipients and/oradditives, such as wetting agents, emulsifying agents, pH bufferingagents, antibacterial and antifungal agents (such as parabens,chlorobutanol, phenol, sorbic acid, and the like). Suitable additivesinclude, but are not limited to, physiologically biocompatible buffers(e.g., tromethamine hydrochloride), additions (e.g., 0.01 to 10 molepercent) of chelants (such as, for example, DTPA or DTPA-bisamide) orcalcium chelate complexes (as for example calcium DTPA orCaNaDTPA-bisamide), or, optionally, additions (e.g. 1 to 50 molepercent) of calcium or sodium salts (for example, calcium chloride,calcium ascorbate, calcium gluconate or calcium lactate). If desired,absorption enhancing or delaying agents (such as liposomes, aluminummonostearate, or gelatin) may be used. The compositions can be preparedin conventional forms, either as liquid solutions or suspensions, solidforms suitable for solution or suspension in liquid prior to injection,or as emulsions. Pharmaceutical compositions according to the presentinvention can be prepared in a manner fully within the skill of the art.

The peptides of the invention, pharmaceutically acceptable saltsthereof, or pharmaceutical compositions comprising these compounds maybe administered so that the compounds may have a physiological effect.Administration may occur enterally or parenterally; for example orally,rectally, intracisternally, intravaginally, intraperitoneally, locally(e.g., with powders, ointments or drops), or as a buccal or nasal sprayor aerosol. Parenteral administration is preferred. Particularlypreferred parenteral administration methods include intravascularadministration (e.g. intravenous bolus injection, intravenous infusion,intra-arterial bolus injection, intra-arterial infusion and catheterinstillation into the vasculature), peri- and intra-target tissueinjection (e.g. peri-tumoral and intra-tumoral injection), subcutaneousinjection or deposition including subcutaneous infusion (such as byosmotic pumps), intramuscular injection, and direct application to thetarget area, for example by a catheter or other placement device.

Where the administration of the peptide is by injection or directapplication, the injection or direct application may be in a single doseor in multiple doses. Where the administration of the compound is byinfusion, the infusion may be a single sustained dose over a prolongedperiod of time or multiple infusions.

The formulations of the pharmaceutical compositions described herein maybe prepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofbringing the active ingredient into association with a carrier or one ormore other accessory ingredients, and then, if necessary or desirable,shaping or packaging the product into a desired single- or multi-doseunit.

It will be understood by the skilled artisan that such pharmaceuticalcompositions are generally suitable for administration to animals of allsorts. Subjects to which administration of the pharmaceuticalcompositions of the invention is contemplated include, but are notlimited to, humans and other primates, mammals including commerciallyrelevant mammals such as cattle, pigs, horses, sheep, cats, and dogs,birds including commercially relevant birds such as chickens, ducks,geese, and turkeys.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in bulk, as a single unit dose, or as a plurality of single unitdoses. As used herein, a “unit dose” is a discrete amount of thepharmaceutical composition comprising a predetermined amount of theactive ingredient. The amount of the active ingredient is generallyequal to the dosage of the active ingredient which would be administeredto a subject or a convenient fraction of such a dosage such as, forexample, one-half or one-third of such a dosage.

The relative amounts of the active ingredient, the pharmaceuticallyacceptable carrier, and any additional ingredients in a pharmaceuticalcomposition of the invention will vary, depending upon the identity,size, and condition of the subject treated and further depending uponthe route by which the composition is to be administered. By way ofexample, the composition may comprise between 0.1% and 100% (w/w) activeingredient.

In addition to the active ingredient, a pharmaceutical composition ofthe invention may further comprise one or more additionalpharmaceutically active agents. Particularly contemplated additionalagents include anti-emetics and scavengers such as cyanide and cyanatescavengers.

Controlled- or sustained-release formulations of a pharmaceuticalcomposition of the invention may be made using conventional technology.

As used herein, “additional ingredients” include, but are not limitedto, one or more of the following: excipients; surface active agents;dispersing agents; inert diluents; granulating and disintegratingagents; binding agents; lubricating agents; sweetening agents; flavoringagents; coloring agents; preservatives; physiologically degradablecompositions such as gelatin; aqueous vehicles and solvents; oilyvehicles and solvents; suspending agents; dispersing or wetting agents;emulsifying agents, demulcents; buffers; salts; thickening agents;fillers; emulsifying agents; antioxidants; antibiotics; antifungalagents; stabilizing agents; and pharmaceutically acceptable polymeric orhydrophobic materials. Other “additional ingredients” which may beincluded in the pharmaceutical compositions of the invention are knownin the art and described, for example in Genaro, ed., 1985, Remington'sPharmaceutical Sciences, Mack Publishing Co., Easton, Pa., which isincorporated herein by reference.

Typically, dosages of the compound of the invention which may beadministered to an animal, preferably a human, range in amount from 1 μgto about 100 g per kilogram of body weight of the animal. While theprecise dosage administered will vary depending upon any number offactors, including but not limited to, the type of animal and type ofdisease state being treated, the age of the animal and the route ofadministration. In one aspect, the dosage of the compound will vary fromabout 1 mg to about 10 g per kilogram of body weight of the animal. Inanother aspect, the dosage will vary from about 10 mg to about 1 g perkilogram of body weight of the animal.

The compound may be administered to an animal as frequently as severaltimes daily, or it may be administered less frequently, such as once aday, once a week, once every two weeks, once a month, or even lessfrequently, such as once every several months or even once a year orless. The frequency of the dose will be readily apparent to the skilledartisan and will depend upon any number of factors, such as, but notlimited to, the type of cancer being diagnosed, the type and severity ofthe condition or disease being treated, the type and age of the animal,etc.

Suitable preparations include injectables, either as liquid solutions orsuspensions, however, solid forms suitable for solution in, suspensionin, liquid prior to injection, may also be prepared. The preparation mayalso be emulsified, or the polypeptides encapsulated in liposomes. Theactive ingredients are often mixed with excipients which arepharmaceutically acceptable and compatible with the active ingredient.Suitable excipients are, for example, water saline, dextrose, glycerol,ethanol, or the like and combinations thereof. In addition, if desired,the vaccine preparation may also include minor amounts of auxiliarysubstances such as wetting or emulsifying agents, pH buffering agents,and/or adjuvants.

The invention also includes a kit comprising the composition of theinvention and an instructional material which describes adventitiallyadministering the composition to a cell or a tissue of a subject. Inanother embodiment, this kit comprises a (preferably sterile) solventsuitable for dissolving or suspending the composition of the inventionprior to administering the compound to the subject.

As used herein, an “instructional material” includes a publication, arecording, a diagram, or any other medium of expression which can beused to communicate the usefulness of the peptide of the invention inthe kit for effecting alleviation of the various diseases or disordersrecited herein. Optionally, or alternately, the instructional materialmay describe one or more methods of using the compositions fordiagnostic or identification purposes or of alleviation the diseases ordisorders in a cell or a tissue of a mammal. The instructional materialof the kit of the invention may, for example, be affixed to a containerwhich contains the multimeric peptide of the invention or be shippedtogether with a container which contains the peptide. Alternatively, theinstructional material may be shipped separately from the container withthe intention that the instructional material and the compound be usedcooperatively by the recipient.

Therapeutic Agents

In other embodiments, therapeutic agents, including, but not limited to,cytotoxic agents, anti-angiogenic agents, pro-apoptotic agents,antibiotics, hormones, hormone antagonists, chemokines, drugs, prodrugs,toxins, enzymes or other agents may be used as adjunct therapies whenusing the multimeric peptide ligand complexes described herein. Drugsuseful in the invention may, for example, possess a pharmaceuticalproperty selected from the group consisting of antimitotic, antikinase,alkylating, antimetabolite, antibiotic, alkaloid, anti-angiogenic,pro-apoptotic agents, and combinations thereof.

Techniques for detecting and measuring these agents are provided in theart or described herein.

Pharmaceutical compositions for use in accordance with the presentinvention may be formulated in conventional manner using one or morephysiologically acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the active ingredients intopreparations which, can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

According to some aspects of the present invention, the method includesproviding to the subject a therapeutic compound in combination with apharmaceutically acceptable carrier.

The present invention further encompasses kits.

Compositions of the present invention may be presented in a pack ordispenser device, such as an FDA approved kit, which may contain one ormore unit dosage forms containing the therapeutic compound as describedherein.

In some embodiments, the kit may include a therapeutic compound (asdescribed herein), metal or plastic foil, such as a blister pack, adispenser device or an applicator, tubes, buffers, and instructions foradministration. The various reagent components of the kits may bepresent in separate containers, or some or all of them may bepre-combined into a reagent mixture in a single container, as desired.The dispenser device or applicator may also be accommodated by a noticeassociated with the container in a form prescribed by a governmentalagency regulating the manufacture, use or sale of pharmaceuticals, whichnotice is reflective of approval by the agency of the form of thecompositions or human or veterinary administration. Such notice, forexample, may be of labeling approved by the U.S. Food and DrugAdministration for prescription drugs or of an approved product insert.

Other embodiments of the invention will be apparent to those skilled inthe art based on the disclosure and embodiments of the inventiondescribed herein. It is intended that the specification and examples beconsidered as exemplary only, with a true scope and spirit of theinvention being indicated by the following claims. While somerepresentative experiments have been performed in test animals, similarresults are expected in humans. The exact parameters to be used forinjections in humans can be easily determined by a person skilled in theart.

Other techniques known in the art may be used in the practice of thepresent invention.

The invention is now described with reference to the following Examples.Without further description, it is believed that one of ordinary skillin the art can, using the preceding description and the followingillustrative examples, make and utilize the present invention andpractice the claimed methods. The following working examples therefore,are provided for the purpose of illustration only and specifically pointout the preferred embodiments of the present invention, and are not tobe construed as limiting in any way the remainder of the disclosure.Therefore, the examples should be construed to encompass any and allvariations which become evident as a result of the teaching providedherein.

Examples

Methods

Mice and Surgical Protocol

All experiments were performed in accordance with the NationalInstitutes of Health and Institutional Animal Care and Use Guidelines.The Animal Care and Use Committee of the University of Virginia approvedall procedures and protocols. C57BL/6 mice from the Jackson Laboratoryand hepcidin knockout mice (HAMP−/−), a kind gift from Dr Cindy Roy(John Hopkins University) were used in this study. All experiments wereperformed on 8-10 weeks old male mice. Kidney ischemia-reperfusionsurgery was performed as previously reported (44). The core temperatureof the mice was maintained between 34° C. and 36° C. using a heatingpad. After anesthetizing the mice (ketamine, 12 mg/ml; atropine, 0.48mg/ml; xylazine, 24 mg/ml; 200 ml mixture administered per 20 g bodyweight), bilateral flank incisions were made, both renal pedicles wereexposed, and cross-clamped for 26 minutes (severe I/R injury) or 24minutes (mild I/R injury). After the determined ischemia, clamps wereremoved, and kidneys were allowed to reperfuse for 24 hours. Ambientpostoperative air temperature was maintained between 30° C. and 32° C.until mice had fully recovered. Sham-operated animals underwentbilateral flank incisions without clamping of renal pedicles.Twenty-four hours later, mice were euthanized, and tissues wereharvested.

Experimental Design

Animals were divided into three groups, injected intraperitoneally withwater and underwent IRI (IRI), injected intraperitoneally with 50-100 μghepcidin at different time points prior to IRI (Hepcidin-IRI) or weresham operated (Sham). The experimental design is depicted in FIG. 2 a.

Blood and Tissue Samples

Before euthanasia, animals were anesthetized and blood was drawn fromthe axilla, following which tissues were harvested. All the tissueslices were fixed with 10% neutral-buffered formalin for paraffinembedding, and with periodate-lysine-paraformaldehyde fixative to befrozen in optimal cutting temperature compound, or snap frozen in liquidnitrogen for subsequent mRNA extraction.

Renal Function

Renal function was determined by measurement of plasma creatinine,determined using a modified Jaffe colorimetric assay (44).

Histologic Examination and Stereologic Analysis of Tissue Morphology

Kidneys were fixed in buffered formalin for 48 h, embedded in paraffin,sectioned (3-μm thickness), and stained with hematoxylin and eosin(H&E). The extent of acute tubular necrosis (ATN) following IRI wasassessed using H&E stained kidney sections. An investigator blinded tothe sample identities was used for these analyses to have an unbiasedapproach. The percentage of tubules in the corticomedullary junctionthat displayed acute tubular necrosis was identified according to thepresence of cast formation, tubule dilation, and/or tubular epithelialde-nucleation.

Serum Iron, Hepcidin and IL-6 Measurement

Serum iron (Abcam), hepcidin (Intrinsic Lifescience) and IL-6 levels(eBioscience) were measured using commercial ELISA kits, as permanufacturers instructions.

Non-Heme Iron Assay

Non-heme iron content in tissue was measured using the method describedin published literature with slight modifications (60). Briefly, tissueswere accurately weighed, finely cut and placed for 20 hours at 65° C. ina solution of 3M HCl/0.61 M trichloroacetic acid. After cooling to roomtemperature, the acid extract was spun at 12,000 rpm to collect thesupernatant. 0.1 mL of the supernatant was incubated with chromogensolution (1.86 mM bathophenanthroline sulfonate/143 mM thioglycollicacid) for 20 minutes, and OD was measured at 535 nm. A standard curvewas generated using an iron standard solution (Ricca Iron AA standard)against water as the blank. Non-heme iron content (μg/g) was calculatedusing the equation described (60).

Flow Cytometry.

Single-cell suspensions from the kidney were prepared using standardmethods. Briefly, kidney section was cut into small pieces andcollagenase (type 2) digested for 20 minutes at 37° C. The digestedkidney was then serially passed through a 100 micron and 50 micron sieveto collect the cell suspension. The cells were then incubated withanti-CD16/32 (Fc block, clone 93; eBioscience, San Diego, Calif.) andstained with APC-eFlour 780-conjugated anti-CD45 (30-F11), PE conjugatedanti-CD11b (M1/70), APC conjugated anti Ly6G (RB6-8C5) and eFlour 450conjugated anti Ly6C (HK1.4). Flow cytometry data were acquired using BDFACSCalibur (BD Biosciences, San Jose, Calif.) with Cytek eight-colorflow cytometry upgrade (Cytek Development, Fremont, Calif.) and analyzedwith FlowJo software 9.0 (Tree Star Inc., Ashland, Oreg.). 100-200,000events/samples were acquired. Neutrophils were identified asCD11b-Ly6G-Ly6C positive cells within CD45 gate.

TUNEL Assay

Apoptotic cells in the kidney were detected by TUNEL assay following themanufacturer's protocol (Roche Diagnostics, Mannheim, Germany). Briefly,PLP-fixed kidney sections were immersed in 3% hydrogen peroxide-methanol(10 minutes) and permeabilized with cold 0.1% Triton-X100 and 0.1%sodium acetate (2 minutes). DNA fragments in apoptotic cells were thenlabeled and identified by terminal transferase dUTP conjugated tofluorescein (Roche Diagnostics) for 60 minutes at room temperature indark. The apoptotic cells were imaged using a Zeiss Axiovert 200microscope with Apo Tome (Zeiss).

Immunohistochemistry.

Kidney sections were embedded in paraffin, 3 μm sectioned, and stainedfor 4 Hydroxynonenal (4-HNE) using standard protocols. Briefly sectionswere deparaffanized in xylenes, rehydrated in a series of ethanol rinsesfrom 100% to 70% ethanol, then washed in distilled water. Sections werethen incubated in 3% H2O2 in methanol for 20 minutes. After treatingwith Avidin and Biotin for 15 minutes each (Vector) the sections wereblocked in blocking buffer containing 10% donkey serum in sodiumphosphate buffer (NaPO4 buffer), at room temperature for 30 minutes.4-HNE antibody was diluted in 1% BSA in NaPO4 buffer (Abcam; 1:1000) andadded to sections overnight at 4° C. Sections were washed 3 times withNaPO4 buffer for 5 minutes each and incubated with biotinylated donkeyanti-goat secondary antibody (Vector) for 1 hour. Sections were washed 3times in NaPO4 buffer, and then incubated in ABC ready-to-use reagent(Vector Labs) for 30 minutes. After another 3 washes with NaPO4 bufferthe sections exposed to diaminobenzidine (DAB) for 5 minutes followed bywashing with in distilled water. The sections were counter-stained with1% methylene blue solution, washed in water and dehydrated with xylene.Sections were imaged by using a Zeiss Axio Imager Z2/Apotome Microscopefitted with motorized focus drives and motorized XYZ microscope stageand integrated to a workstation running StereoInvestigator software,version 10.51 (MBF Bioscience, Williston, Vt.)

Immunofluorescence

Three micron paraformaldehyde/lysine/periodate (PLP) fixed kidneysections were used for the immunofluorescence detection of neutrophilsand CD11b cells. Briefly, the kidney sections were air dried, andtreated with 0.3% triton X in 10% horse serum for 30 minutes. Afterwashing the sections with PBS, anti CD16/32 antibody was added to blockthe FC receptors. This was followed by incubation with FITC-labeledanti-neutrophil antibody (7/4, Cederline, 1 in 30) and PE-labeled CD11b(M1/70, ebioscience, 1 in 30) diluted in 10% horse serum for 1.5 hours.The sections were then washed 3 times in PBS and mounted with ProLongGold anti fade agent with DAPI (Life Technologies). Sections were imagedby Zeiss Axiovert 200 microscope with Apo Tome (Zeiss)

Cell Culture

A mouse proximal tubule cell line (TKPTS cells, kindly provided by Dr.E. Bello-Reuss, University of Texas Medical Branch, Galveston, Tex.) wasused in these studies. The cells were cultured in advanced DMEM/F12medium supplemented with glutamine, 7.5% FBS and antibiotics. Cells weregrown to 80% confluence and maintained at 37° C. in 5% CO₂. Allexperiments were performed in serum free DMEM/F12 medium for a maximumof 24 hours.

In-Vitro Hypoxia and Re-Oxygenation

TKPTS cells were subjected to hypoxia using a hypoxic chamber (StemcellTechnologies) for 6 hrs (5% CO₂, 1% O₂, and 94% N₂) followed byre-oxygenation for 6 hrs (5% CO₂, 21% 02, and 74% N₂). Three cultureconditions were set up: 1. Normoxia (which served as the base linecontrol); 2. Hypoxia; and 3. Cells pretreated with 50 μM hepcidin andsubjected to hypoxia. Experiments were carried out in serum freeadvanced DMEM/F12 medium. At the end of re-oxygenation, cells werestained with Annexin V/PI and extent of apoptosis was determined usingflow cytometry.

Iron Stimulation of Splenocytes

Splenocytes were harvested from 10-week-old B6 male mice. After excisingthe spleen, red blood cells were lysed and the remaining splenocyteswere seeded at 1×106 cells/mL in 24-well tissue culture plates andcultured under 5% CO2 at 37° C. for 24 hours. The culture mediumconsisted of RPMI 1640 medium supplemented with 10% fetal calf serum(FCS), 100 U/mL penicillin, 100 μg/mL streptomycin, 1% non-essentialamino acids (Gibco/Life Technologies, Foster, Calif., USA), 2 mML-glutamine (Sigma), and 50/μM 2-mercaptoethanol. After resting for 24hours, the cells were treated with 10 μM ferric ammonium chloride (FAC),1 μg/mL hepcidin or saline for 4 hours. The cell culture supernatantswere harvested and quantified for the levels of IL-6 by ELISA(eBioscience).

Western Blot Analysis

Snap frozen tissue sections were homogenized in Tris-Triton tissue lysisbuffer containing complete protease inhibitor cocktail using a DounceHomogenizer. Membrane fractions were isolated from the whole kidneylysate using membrane isolation kit (Thermo Pierce) to determineferroportin expression. H-Ferritin was measured in the whole kidney andspleen lysates. Protein content in the homogenate was estimated usingthe BCA protein estimation kit. Twenty to thirty microgram of proteinper sample were loaded on a 10% NuPage Bis-Tris gel under reducingconditions. The resolved proteins were transferred onto a Nitrocellulosemembrane (Licor Bioscience) and probed with goat anti-mouse H-Ferritin(Santa Cruz) and rabbit anti-mouse ferroportin (Novus Biologics)antibodies. The primary antibodies were detected with Donkey anti-goatAlexa 800 and goat anti-rabbit Alexa 800 antibodies (LiCor Bioscience).Mouse monoclonal GAPDH and β-Actin (Abcam) were used as the loadingcontrols and detected using Donkey anti Mouse Alexa 680 antibody (LiCorBioscience). Quantitation of data was performed using densitometrysoftware (Licor Bioscience).

Real Time PCR

For RNA isolation, frozen tissues were re-suspended in RLT buffer(Qiagen Inc, Valencia, Calif.) and homogenized using TissueLyser system(Qiagen Inc, Valencia, Calif.). Total RNA from tissue homogenate waspurified using RNAeasy mini kit (Qiagen Inc, Valencia, Calif.) followingmanufacturers instructions. 1 μg of RNA was used to synthesize cDNAusing the iScript cDNA synthesis kit (Bio-Rad). The cDNA template wasmixed with iTAQ SYBR green universal super mix (Bio-Rad) andquantitative PCR was carried out on CFX Connect system (Bio-Rad).Predesigned primers for Caspase-3 and Bcl-2 were purchased from Bio-rad.Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was amplified inparallel and used as the reference gene in quantification. Data isexpressed as the relative gene expression and was calculated using the 2(−ρρC(T)) method.

Statistics.

GraphPad Prism 6 (GraphPad Inc.) was used to analyze and present thedata. Data were analyzed, after transformation if needed to generate anormal distribution, by t test or 1-way ANOVA with post hoc analysis asappropriate. P<0.05 was used to indicate significance.

Hepcidin Agonist PR73 (Mini-Hepcidin)—

This 10 amino acid peptide agonist of Hepcidin was used in animals ateither 100 or 200 nmol. Its description and sequence can be found inWO2013086143 A1 (Ganz et al.). The effects on plasma creatinine levelswere determined.

Results

Hepcidin Prevents Renal Ischemia-Reperfusion Induced Iron Dyshomeostasis

There is a dynamic and continuous exchange of iron between hepatospleniccompartments and the plasma. This process is regulated by hepcidinthrough its modulation of reticuloendothelial (splenic red pulpmacrophages and hepatic Kupffer cells) ferroportin expression. Whileprior studies have demonstrated increased serum iron levels followingrenal IRI, no previous studies have characterized the dynamic changes iniron distribution between different organ and tissue compartmentsfollowing renal IRI. Here, we first confirmed the previous observationsthat renal IRI triggers a significant increase in serum iron levelscompared to the sham operated animals (FIG. 1a , p<0.0005). Hepcidintreatment significantly reduced IRI-induced increase in serum ironlevels (FIG. 1a , p<0.001). We next measured non-heme iron levels in thekidney, spleen and liver of the animals. IRI was associated with asignificant increase in kidney non-heme iron content compared to shamoperated animals (FIG. 1b , p<0.001) and hepcidin treatmentsignificantly prevented IRI-induced kidney iron accumulation (FIG. 1b ,p<0.05). Next, we determined the non-heme iron content in the spleen andliver of the three experimental groups. Hepcidin treated mice had asignificantly higher splenic non-heme iron content than IRI or shamgroups (FIG. 1c , p<0.001 and <0.05 respectively). In IRI group, livernon-heme iron levels were significantly less than the sham group (FIG.1d , p<0.0005) and hepcidin treatment effectively prevented IRI-induceddepletion of liver non-heme iron (FIG. 1d , p<0.0001).

Increase in serum iron levels following renal IRI raised the questionwhether this would result in an iron-dependent increase in hepcidinsynthesis and release. We, therefore, measured hepcidin expression(transcripts and protein) in the liver and serum of the threeexperimental groups of mice. Renal IRI resulted in a significantupregulation of liver hepcidin gene expression compared to sham mice(FIG. 1e , p<0.005). Hepcidin treatment before IRI significantlyprevented the IRI-induced upregulation of hepcidin with gene expressionlevels comparable to sham operated animals (FIG. 1e , IRI Vs.Hepcidin-IRI, p<0.005). Serum hepcidin paralleled these changes withlevels significantly elevated following IRI in untreated mice ascompared to sham (FIG. 1f , IRI Vs Sham, p<0.0001) or hepcidin-treatedmice (FIG. 1f , IRI Vs Hepcidin-IRI, p<0.0001). Hepcidin levels were notsignificantly different between hepcidin-treated and sham groups.

Collectively, our observations indicate that IRI induces dynamic changesin iron metabolism characterized by hepatosplenic iron depletion,systemic iron mobilization, kidney non-heme iron accumulation andpost-IRI hepcidin induction. Hepcidin treatment before IRI effectivelyprevents these changes.

Hepcidin Mitigates IRI, Reduces ATN and Improves Renal Function

As iron plays an important role in renal IRI, next, we examined thefunctional significance of hepcidin-mediated modulation of ironhomeostasis on IRI. Compared to sham, plasma creatinine of untreated IRImice was significantly higher (FIG. 2b , p<0.0001). A single bolus of 50or 100 μg hepcidin (intraperitoneally), 24 hours before IRI,significantly lowered plasma creatinine levels (FIG. 2b , p<0.0001). Asthere were no significant differences in plasma creatinine of miceinjected with either 50 or 100 □g hepcidin, all further experiments wereperformed using 50 μg hepcidin dose.

We next established the timing of hepcidin injection that would offermaximum protection against IRI. We injected mice with either hepcidin orwater at 2 or 8 hours prior to ischemia and measured plasma creatinine24 hours after reperfusion. Remarkably, hepcidin injection at both 2 and8 hours prior to IRI significantly reduced plasma creatinine levels(FIG. 2b , p<0.001 compared to untreated IRI mice). However, maximumprotection was observed when hepcidin was injected 24 hours before IRI(FIG. 2b ). Hence, in all further experiments, hepcidin was injected 24hours before IRI.

The protective role of hepcidin was also evidenced by a better-preservedrenal architecture that correlated well with the creatinine levels.Following IRI, kidney sections of untreated mice showed extensivetubular necrosis all the way from the S3 segment in the outer strip ofthe outer medulla and extended to the S1 and S2 segment of the proximaltubule in the deep cortex (FIG. 2d , Sup FIG. 1b ). However,hepcidin-treated mice had fewer necrotic tubules and luminal debris inthe 51 and S2 segments of the proximal tubules and exhibited normalepithelial morphology. The S3 segment of the outer medulla alsodemonstrated substantially reduced tubular damage and less evidenttubular casts (FIG. 2e , Sup FIG. 1c ). The preserved renal function andarchitecture was associated with significant reduction in kidney injurymarker neutrophil gelatinase-associated lipocalin (NGAL). Renal IRIresulted in a significant increase in NGAL (Sup FIG. 1e , IRI Vs Sham,p<0.0001). In contrast, the hepcidin-treated IRI mice had significantlylower expression of NGAL (Sup FIG. 1d , IRI Vs Hepcidin-IRI, p<0.001).Collectively, these findings demonstrated a protective role of hepcidinin renal IRI.

Hepcidin Decreases Ischemia-Induced Renal Epithelial Apoptosis.

Apoptosis is a major mediator of tubular cell death following renal IRI(33) and iron can trigger renal epithelial apoptosis. To investigate ifhepcidin-mediated protection in renal IRI is mediated through modulationof epithelial apoptosis, we measured apoptosis-related genes in kidneysof the 3 experimental groups. Compared to sham operated mice, Caspase-3was significantly upregulated in the kidneys of untreated IRI mice (FIG.3a , p<0.0001). Hepcidin treatment significantly reduced IRI-inducedCaspase-3 expression in the kidneys (FIG. 3a , p<0.005). Interestingly,Caspase-3 expression in the kidneys of hepcidin treated mice was evenlower than the sham mice (FIG. 3a , Hepcidin-IRI Vs Sham, p<0.03).Expression of Bcl-2, an anti-apoptotic gene, with known protectiveproperties in renal IRI (34), was also different in the three groups.Bcl2 expression was significantly reduced in IRI mice compared to sham(FIG. 3b , p<0.0008) and hepcidin-IRI mice had a significantly higherexpression of Bcl2 compared to untreated IRI mice (FIG. 3b , p<0.0008).To further establish the observed reduction in apoptosis in thehepcidin-IRI mice, we stained the kidney sections with terminaldeoxynucleotidyl transferase-mediated digoxigenin-deoxyuridine nick-endlabeling (TUNEL) as an indicator of apoptosis. We did not detect anyTUNEL-positive cells in kidney sections of sham-operated mice (data notshown). As expected, 26 minutes of renal ischemia followed by 24 hoursof reperfusion resulted in a significant increased in TUNEL reactivityin the nuclei of the renal tubules (FIG. 3c-d ). In contrast,hepcidin-IRI mice had remarkably fewer TUNEL-positive tubules in thekidney (FIG. 3e-f ).

To investigate if hepcidin has a direct anti-apoptotic effect on renaltubular epithelial cells, we subjected murine proximal renal tubularcells (TKPTS) to hypoxia-reoxygenation injury in vitro (34). TKPTS cellswere first treated with or without 50 μM hepcidin for 24 hours in 5%serum containing medium. After transferring to serum free medium, thecells were then cultured in 1% hypoxia for 6 hours. Cells cultured underidentical but normoxic conditions were used as controls. After 6 hoursof re-oxygenation, cells were stained for Annexin V/PI to measure theextent apoptosis/necrosis induced by the hypoxic-reoxygenation insult.Hypoxia led to a significant increase in Annexin V/PI positive cellscompared to normoxic condition (FIG. 3g-h , p<0.005). Howeverhepcidin-treated cells subjected to hypoxia demonstrated significantlylower Annexin V/PI staining compared to untreated cells (FIG. 3g-h ,p<0.005) and the frequency of cells undergoing apoptosis were reduced tolevels seen under normoxia (FIG. 3g-h ). These findings reinforce ourin-vivo observations and suggest a direct anti-apoptotic role ofhepcidin.

Hepcidin Treatment Reduces Renal IRI-Induced Oxidative Stress andInflammation.

Oxidative stress-induced apoptotic cell death is believed to be one ofthe major pathways involved in the pathogenesis of kidney I/R injury(35, 36). Iron is known to play central player in triggering oxidativetissue injury through its ability to redox cycle and generate oxidativeradicals such as hydroxyl (OH). Iron chelation with desferrioxamine andantioxidants such as superoxide dismutase (SOD) attenuate IRI (37-39).Since hepcidin treatment directly modulates IRI-associated irondyshomeostasis, we examined whether the reduced injury observed withhepcidin treatment is associated with lowering of oxidative stress.4-Hydroxynonenal (4-HNE), an aldehyde product of membrane lipidperoxidation is produced by oxidative stimuli and is conventionally usedas a tissue marker of oxidative stress (40). Kidneys of untreated IRImice showed prominent 4-HNE immunoreactivity, indicative of lipidperoxidation and oxidative injury (FIG. 4a (top panel), and Sup FIG. 2a). The pattern of staining mirrored renal injury distribution, beingmost intense in the medulla and spreading out with lesser intensity inthe corticomedullary junction and deep cortex. As compared to theuntreated mice, kidneys of hepcidin-treated mice showed far less 4-HNEimmunoreactivity. Only a restrictive 4-HNE staining could be detected insome regions of the deep medulla and corticomedullary junction (FIG. 4a(bottom panel) and Sup FIG. 2b ). Further, the intensity of staining wassignificantly less compared to the untreated mice. Taken along with theeffect of hepcidin in preventing renal iron accumulation, these findingssupport the inhibitory effect of hepcidin treatment on renal IRI-inducedoxidative stress.

Apoptosis and ROS generation are early events following renal IRI and isfollowed by the infiltration of immune cells which leads to theamplification of kidney pathology (33, 35, 6). Multiple previous studieshave linked the infiltration of immune cells with renal pathologyfollowing IRI (41, 42). Therefore we investigated whether the reducedapoptosis and ROS seen with hepcidin treatment is associated withdecreased kidney infiltration of inflammatory cells. Compared to shamoperated mice, IRI in untreated mice resulted in a large and significantincrease in the frequency of kidney CD45+ cells (FIG. 4b-c , p<0.005).However, hepcidin treatment reduced renal inflammatory cell infiltrationby almost half (FIG. 4b-c , p<0.005). Prior studies have shown thatneutrophils comprise the majority of immune cells that infiltrate thekidneys following I/R injury (41). We identified neutrophils asCD45⁺CD11b^(hi)Ly6G⁻Ly6C^(hi) cells. As expected, neutrophils were themost abundant immune cells in the kidneys following IRI (FIG. 4c ,p<0.005). Hepcidin treatment significantly reduced IRI-induced renalneutrophil inflammation compared to the untreated IRI mice (FIG. 4c ,p<0.005). Collectively, these observations demonstrate that hepcidintreatment reduces renal IRI-associated inflammation.

Hepcidin Preserves Cytoprotective H-Ferritin by Preventing IRI-InducedRenal Ferroportin Induction.

The primary known function of hepcidin is to induce ferroportindegradation and increase intracellular iron stores. In turn, thisresults in the induction of H-Ferritin, an iron binding ferrooxidasewith cytoprotective function (43). To address if the protection observedin our studies is due to hepcidin-induced ferroportin degradation andincreased H-Ferritin, we measured the expression of these proteins inkidneys of sham, IRI and hepcidin-IRI mice. Ferroportin expression wasincreased in the kidneys of IRI mice compared to sham mice (FIG. 5a-b ,p<0.05). Pretreatment with hepcidin maintained ferroportin expression(and thus maintain intracellular iron levels) at levels comparable tosham mice (FIG. 5a-b , p<0.01). Correlating with these changes inferroportin (expected to induce intracellular iron depletion),H-Ferritin levels were significantly lower in the kidneys following IRI(FIG. 5c-d ). Hepcidin treatment was associated with significantlyhigher H-Ferritin expression compared to both untreated IRI mice andsham mice (FIG. 5c-d , hepcidin-IRI Vs IRI p<0.005, hepcidin-IRI Vs Shamp<0.05). Collectively, our data demonstrates that renal IRI isassociated an increase in kidney ferroportin and decrease in kidneyH-Ferritin and hepcidin treatment prevents these changes.

Hepcidin Deficiency Increases Susceptibility to AKI

Given the significance of hepcidin-mediated ferroportin regulation inpreventing renal IRI, we hypothesized that genetic hepcidin deficiencywould aggravate IRI. Here, we demonstrate that hepcidin knock out mice(HAMP^(−/−)) develop severe renal injury compared to hepcidin sufficient(WT) controls following IRI. To demonstrate this, we first subjected theHAMP^(−/−) and WT mice to a milder degree of IRI (24 minutes ischemia,24 hours reperfusion). There was no significant rise in plasmacreatinine after mild IRI in the WT mice and sham operated HAMP^(−/−)mice (FIG. 6a ). However, the plasma creatinine of HAMP^(−/−) micesubjected to IRI was significantly elevated (FIG. 6a , p<0.0008). Renalhistology further corroborated the deleterious effect of hepcidindeficiency in AKI. There were higher numbers of casts and necrotictubules in the S1, S2 and S3 segments in HAMP^(−/−) kidneys after IRI(FIG. 6b , and Sup FIG. 3b ). To investigate if hepcidin re-constitutioncould rescue HAMP^(−/−) mice from IRI, we injected these mice with asingle 50 μg bolus of hepcidin, 24 hours before renal ischemia andsacrificed them 24 hours after reperfusion. Strikingly, a singleinjection of hepcidin afforded an almost complete protection against IRIin the HAMP^(−/−) mice. Plasma creatinine levels of hepcidinreconstituted HAMP^(−/−) mice were significantly lower compared tountreated mice with levels comparable to that in sham-operated mice(FIG. 6a , p<0.0007). There was also a remarkable preservation of renalhistology (FIG. 6c , and Sup FIG. 3c ) in the hepcidin treatedHAMP^(−/−)-IRI mice.

While renal IRI caused a significant increase in NGAL (Sup FIG. 3d ,HAMP^(−/−)-IRI Vs Sham, p<0.005), hepcidin treatment significantlylowered NGAL expression in HAMP^(−/−)-IRI mice compared to untreatedHAMP^(−/−)-IRI mice (Sup FIG. 3d , HAMP^(−/−)-IRI VsHAMP^(−/−)-Hepcidin-IRI, p<0.05).

As HAMP−/− mice exhibit chronic iron overload, we investigated ifprotection induced by reintroduction of hepcidin was associated withchanges in systemic iron levels. We measured the serum iron and non-hemeiron content in the kidney, spleen and liver of sham, untreated andhepcidin injected HAMP^(−/−) mice after IRI. Compared to sham mice,there was a paradoxic trend toward lowering of serum iron in HAMP^(−/−)mice after IRI, However, hepcidin reintroduction significantly loweredserum iron in HAMP^(−/−)-IRI mice compared to both the HAMP−/−-IRI andsham mice (FIG. 6e , HAMP^(−/−)-hepcidin-IRI Vs untreatedHAMP^(−/−)-IRI, p<0.005, HAMP^(−/−)-hepcidin-IRI Vs sham, p<0.0005). Thelowering of serum iron in hepcidin treated HAMP^(−/−)-IRI mice wereassociated with increased hepatic sequestration of non-heme iron as theliver non-heme iron content of hepcidin-treated HAMP^(−/−)-IRI mice wassignificantly higher than the untreated HAMP^(−/−)-IRI mice (FIG. 6f ,p<0.005) and sham controls (FIG. 6f , p<0.0005). While Prussian bluestaining demonstrated qualitative increases in splenic iron content inhepcidin-treated HAMP^(−/−) mice, we could not detect quantitativedifferences in the non-heme iron content of the spleen and kidneys ofsham, untreated and hepcidin injected HAMP^(−/−) mice after IRI (datanot shown). Taken together, our data shows that hepcidin deficiencysignificantly aggravates kidney's susceptibility to IRI and thatreconstitution with exogenous hepcidin and restoration of ironhomeostasis can mitigate IRI.

Hepcidin Reconstitution Preserves H-Ferritin and Prevents Apoptosis.

WT mice injected with hepcidin had higher levels of H-Ferritin and lessapoptotic kidney injury. To investigate if the kidneys of hepcidinreconstituted HAMP-/mice shared similar features as their WTcounterparts after IRI, we quantified H-Ferritin expression by westernBlot. We also measured apoptotic pathway markers, Caspase-3 and Bcl-2expression by RTPCR, and stained the kidneys for TUNEL-positive cells.Consistent with the observation in WT mice, H-Ferritin levels weresignificantly decreased following IRI in HAMP^(−/−)-IRI mice compared totheir sham counterparts (FIG. 7a-b , p<0.0005). In contrast, in hepcidintreated HAMP^(−/−)-IRI mice, H-Ferritin levels were significantly higherthan in untreated HAMP^(−/−)-IRI mice (FIG. 7a-b , p<0.05) but lowerthan in sham mice (FIG. 7a-b , p<0.005). As H-Ferritin is known to exertanti-apoptotic effects under settings of IRI (43), we measured apoptosisrelated genes and indicators. Caspase-3 was significantly upregulated inthe untreated HAMP^(−/−)-IRI mice as compared to sham mice (FIG. 7c ,p<0.007). Remarkably, IRI-induced upregulation of Caspase-3 was almostcompletely prevented by a single injection of hepcidin in HAMP^(−/−)-IRImice with levels comparable to that in sham mice (FIG. 7c ,HAMP^(−/−)-IRI Vs HAMP^(−/−)-Hepcidin-IRI, p<0.007). We also measuredthe expression of anti-apoptotic gene Bcl-2. Expression of Bcl-2 (d) wassignificantly lower in the HAMP^(−/−)-IRI mice compared to sham mice(FIG. 7d , p<0.05). Hepcidin treatment prevented the decrease in Bcl-2expression (FIG. 7d , HAMP^(−/−)-Hepcidin-IRI Vs HAMP^(−/−) -IRI,p<0.005). Corresponding to these changes, in comparison withHAMP^(−/−)-IRI mice, there were significantly fewer TUNEL-positive cellsin the kidney of hepcidin treated HAMP^(−/−)-IRI mice (FIG. 7g , SupFIG. 4c ), especially in the corticomedullary region (FIG. 7e , Sup FIG.4c ). We did not detect any TUNEL positive cells in the kidneys of shamoperated HAMP^(−/−) mice (FIG. 7e , Sup FIG. 4a ). Collectively, thesefindings demonstrate that renal IRI in the setting of hepcidindeficiency is associated with worse outcome, is associated withreduction in cytoprotective H-Ferritin, and an increase in epithelialapoptosis. Hepcidin reconstitution preserves H-Ferritin and preventsapoptosis.

Hepcidin Reconstitution Restores Protection Against IRI-InducedOxidative Stress and Inflammation in HAMP−/− Mice.

To evaluate the role of ROS in mediating apoptosis in the HAMP^(−/−)mice, we stained the kidneys for 4-HNE immunoreactivity. The kidneys ofsham operated HAMP^(−/−) mice did not demonstrate any 4-HNEimmunoreactivity (FIG. 8a ). In comparison, kidneys of untreatedHAMP^(−/−)-IRI mice showed high immunoreactivity for 4-HNE adduct (FIG.8b ). 4-HNE staining could be detected in region of the papilla, deepmedulla and along the corticomedullary junction. However, hepcidinreconstituted animals demonstrated far less immunoreactivity to 4-HNE,with only a faint speckled pattern observed in the papilla and deepmedulla (FIG. 8c ). These observations suggest that protection mediatedby hepcidin reconstitution in HAMP−/− mice is associated with reducedIR-induced oxidative stress.

The lack of apoptosis and ROS in hepcidin treated HAMP^(−/−)-IRI micewere associated with reduced inflammatory infiltration in the kidneys.Sham operated HAMP^(−/−) mice did not show any neutrophils and CD11bcell infiltration (data not shown). A large number of neutrophils (7/4antigen positive cells) could be detected in the kidneys of untreatedHAMP^(−/−) mice after IRI (FIG. 8d ). They were distributed around theinjured tubules and in the corticomedullary region. Similarly, therewere large numbers of CD11b+ cells surrounding the injured tubules (FIG.8f ). However, hepcidin reconstitution in HAMP^(−/−)-IRI mice almostcompletely prevented kidney injury-associated neutrophil or CD11b cellinfiltration (FIGS. 8e and g respectively). The reduction ininflammation correlated well with the lack of apoptosis and ROS in theprotected animals.

Hepcidin Prevents IRI-Induced Ferroportin Upregulation and Splenic IronDepletion.

Role of spleen in the pathophysiology of renal IRI is now wellestablished (44). As splenic iron content decreased following renal IRIwith an associated increase in serum iron, we stained the spleens ofboth WT and HAMP^(−/−) mice for ferroportin, the only known ironexporter. Renal IRI resulted in an upregulation of splenic ferroportinin both WT and HAMP^(−/−) mice. Ferroportin expression co-localizedmostly with F4/80+ red pulp macrophages (FIG. 9a middle panel). Hepcidintreatment induces a dramatic decrease in surface expression offerroportin (FIG. 9a bottom panel). In sham operated HAMP^(−/−) mice,because of chronic hepcidin deficiency, basal splenic ferroportinexpression was much higher than in the WT mice (FIG. 9a top panel) andas expected, ferroportin expression was not further increased by IRI.Hepcidin reconstitution induced a lower ferroportin expression thansham-operated mice. As hepcidin-induced ferroportin degradation isexpected to increase intracellular iron levels, we hypothesized thatthis would lead to an increase in H-Ferritin synthesis. Indeed,hepcidin-treated IRI mice had significantly higher H-Ferritin comparedto both untreated IRI mice and sham controls (FIG. 9b WT hepcidin IRI vsIRI, Hepcidin IRI vs Sham, p<0.05) and (FIG. 9c HAMP^(−/−)-Hepcidin-IRIvs HAMP^(−/−)-IRI, HAMP^(−/−)-Hepcidin-IRI vs HAMP^(−/−) Sham,p<0.005)}. Thus, renal IRI results in an increase in splenic ferroportinexpression, and hepcidin treatment prevents IRI-induced ferroportinupregulation. Hepcidin mediated ferroportin degradation leads to spleniciron accumulation and consequent increase in H-Ferritin synthesis (whichwill reduce free iron levels).

Iron and Hepcidin have Contrasting Effects on Interleukin-6 (IL-6)Production

Interleukin-6 (IL-6) is a pleiotropic cytokine and its signaling pathwayhave been linked with the pathogenesis of renal IRI (45, 46). Toinvestigate if hepcidin-mediated protection in renal IRI was associatedwith changes in IL-6, we measured the serum IL-6 levels by ELISA.Compared to sham operated mice, renal IRI resulted in a significantincrease in serum IL-6 in both untreated WT (FIG. 10a WT IRI Vs WT Sham,p<0.007) and HAMP^(−/−) mice (FIG. 10b HAMP^(−/−)-IRI Vs HAMP^(−/−)Sham, p<0.05). Hepcidin treatment prevented IRI-induced increase in IL-6levels in both WT (FIG. 10a WT-IRI Vs WT Hepcidin-IRI, p<0.007) andHAMP^(−/−) mice (FIG. 10b HAMP^(−/−)-IRI Vs HAMP^(−/−)-Hepcidin-IRI,p<0.05), with levels comparable to those in sham-operated mice. Toexplore the possibility that free iron might directly regulate IL-6production in immune cells, we treated splenocytes from WT mice withferrous sulfate and measured the IL-6 secretion into supernatants byELISA. Untreated splenocytes and hepcidin-treated splenocytes were usedas controls. There was no significant difference in IL-6 levels betweenthe untreated splenocytes and hepcidin-treated cells (FIG. 10c ).However treatment with iron significantly increased splenocytes IL-6secretion compared to both untreated and hepcidin-treated cells (FIG.10c , p<0.005). These findings demonstrate that excess splenocyte freeiron leads to increased production of IL-6, a cytokine implicated in thepathogenesis of renal IRI.

Minihepcidin (PR-73) Prevents AKI Following IRI.

Animals were subjected to IRI with or without treatment with thehepcidin agonist at 100 or 200 nmol. The effects at 200 nmol arestatistically significant in reducing plasma creatinine levels relativeto the other groups.

Discussion

In this study, we demonstrate that supplementing mice with exogenoushepcidin protects the kidney from IRI as manifested by an improved renalstructure and function. First, we present evidence that renal IRIresults in a systemic iron mobilization from the hepatospleniccompartments and is associated with kidney iron accumulation. Hepcidintreatment prevents IRI-induced iron dyshomeostasis by inducingferroportin degradation, H-Ferritin induction and effectivehepatosplenic sequestration of iron. These effects are associated withreduced ROS, apoptosis and inflammation in the kidney. We also provideevidence that genetic hepcidin deficiency severely increasessusceptibility to renal IRI and that hepcidin reconstitution restoresprotection. Lastly, our in-vitro data provides evidence for additionalmechanisms of hepcidin's protective effects: 1) a direct anti-apoptoticeffect of hepcidin on renal epithelial cells and 2) an anti-inflammatoryeffect to counter iron-induced IL-6 production in resting splenocytes.

The implication of iron in pathogenesis of AKI in absence of systemiciron overload is a new and expanding field. Iron plays a fundamentalpathological role in IRI and iron chelation with desferrioxamine hasbeen reported to provide protection and significantly improve itsseverity and outcomes (7, 37). The beneficial effect of iron-chelatingagents against renal IRI may be a result of binding (and, therebyremoval) of iron from both intra and extracellular space (37, 47), andin the case of apotransferrin (15) and in the case of NGAL (48), thedelivery of iron to viable cells to enhance recovery. Our currentfindings not only corroborate the role of iron in IRI but also indicatethe importance of extra-renal iron homeostasis in renal pathophysiology.

The protective property of H-Ferritin is well established in AKI as waselegantly by a recent study that demonstrated worse outcome of AKIfollowing deletion of H-Ferritin from renal proximal tubules (49).Anti-apoptotic property of H-Ferritin has also been demonstrated in aliver ischemia model, where H-Ferritin was shown to suppress theactivation of the transcription factor NFκB in a HO-1 independent manneras well as to have a direct anti-apoptotic effect on hepatocytes (43).Each molecule of H-Ferritin can to bind to 4500 Fe²⁺ ions (50) making itan important endogenous iron chelator. This sequestration of Fe²⁺ byH-Ferritin prevents it from participating in the Fenton reaction andthus inhibits iron-mediated pro-oxidant activity. The reduction in ROSand hence apoptosis has been attributed to reduced availability of ironfollowing IRI (7, 8-17-18). In our study, hepcidin treatment increasedrenal H-Ferritin levels after IRI. Hepcidin also preserved kidneyH-Ferritin in HAMP^(−/−) animals. Thus, by limiting the availability offree intracellular iron in the renal cells, H-Ferritin could potentiallyreduce renal ROS and ensuing apoptosis. Actions of hepcidin on thespleen are equally if not more important due to its large iron content.Hepcidin induced splenic ferroportin degradation, causing splenic ironsequestration and H-Ferritin induction after renal IRI. It can thus beargued that reduced splenic ferroportin and increased splenic H-Ferritinprevented systemic iron mobilization following IRI and limited itsavailability to catalyze ROS production in the kidney and prevent tissueinjury. A direct anti-apoptotic role of hepcidin on renal epithelialcells as observed in our in-vitro hypoxia-reoxygenation studies islikely to serve as an additional protective mechanism. Additionalstudies are required to define the mechanisms of hepcidin'santi-apoptotic effects.

Recent studies have suggested an important role for liver and spleen(51, 44) in the pathophysiology of renal IRI. In this study, wedemonstrate that IRI induces hepatosplenic iron export, which likelycontributes to the pathogenesis of renal IRI. This is not surprising assplenic macrophages along with hepatocytes are the main storage sitesand sources of iron in the body (52). Because hepcidin acts rapidly onthe splenic macrophages (53), under our experimental conditions,contributions from the spleen are likely to be more critical. Theability of spleen to sequester iron has been observed in LPS-inducedinflammation (54). It was also demonstrated that following endotoxemiaand inflammation, hepatic hepcidin production increases. A model ofliver IRI similarly showed that serum and hepatic hepcidin increasesafter IRI (55). Our observation that renal IRI induces hepatic hepcidinexpression to increase serum levels further confirms these studies.Thus, under settings of both sterile (IRI) and non-sterile acuteinflammation, body's physiological response seems to inducehepcidin-dependent intracellular iron sequestration to protect theorganism from free iron-mediated injury. Paradoxic increase in splenicferroportin despite an increase in hepcidin levels after IRI needsfurther study. It could be mediated by signals from the injured kidneyindependent of hepcidin.

Our data in the HAMP^(−/−) mice does not completely mimic the findingsin WT mice, where the serum iron levels actually decreased and liverlevels increased after renal IRI. However, it should be noted that thisis a model of constitutive iron overload and spleens of HAMP^(−/−) miceexpress high levels of cell surface ferroportin and are severely irondepleted even under normal conditions (56). Therefore, unlike WT mice,where spleen contributes significantly to systemic iron mobilizationafter IRI, a similar phenomenon is not observed in the HAMP^(−/−) mice.Increased hepatic non-heme iron in HAMP^(−/−) mice following IRIsuggests hepcidin-independent mechanisms and needs further study.

CD45+ immune cells start infiltrating the kidney early afterreperfusion, with neutrophils playing a major role in the ensuingpathological response (6, 41). Hepcidin treatment reduced theinfiltration of total CD45+ cells majority of which were neutrophils.Whether hepcidin affects the chemotaxis of immune cells to the kidney orthe reduction in infiltration is a consequence of less epithelial injuryneeds further investigation. Examining early events preceding theinfiltration of cells may provide clues to this question and warrantfurther investigation.

Inflammatory cytokines such as IL-6 have been shown to be deleterious onthe outcome of renal IRI (45, 46) and are made locally as well as byinfiltrating macrophages (57). Interestingly, IL-6 is also a powerfulinducer of hepcidin (58). Hepcidin treatment significantly preventedIRI-induced increase in splenic, renal, and systemic IL-6 levels. Thesource of systemic IL-6 was not investigated in this study. As systemiciron levels are increased after renal IRI, we examined whether ironcould directly regulate IL-6 production. Our in-vitro data suggests thatiron-dependent increase in splenic IL-6 secretion could be an importantsource. Our in-vitro observations are consistent with findings byDomenico et al. (59) who showed that hepcidin pretreatment of ironloaded bone marrow macrophages reduced LPS-induced IL-6 and TNF alphaproduction. Collectively, our observations suggest that renalIRI-induced reduction in splenic H-Ferritin and increase in free ironcould induce splenocytes to secrete IL-6.

Our studies demonstrate a novel renal protective effect of hepcidin. Wepropose that hepcidin protects the kidney from IRI by acting not only onthe renal epithelial cells but also through its effects on extra-renaliron homeostasis, with spleen being a dominant player. Hepcidin'sprotection is achieved mainly by inducing ferroportin degradation andsubsequent induction of H-Ferritin, which would lead to a safesequestration of free iron. Further studies are required to investigatethe relative contribution of renal and extra-renal ferroportin inmediating hepcidin's protective effects. The disclosures of each andevery patent, patent application, and publication cited herein arehereby incorporated by reference herein in their entirety.

Headings are included herein for reference and to aid in locatingcertain sections. These headings are not intended to limit the scope ofthe concepts described therein under, and these concepts may haveapplicability in other sections throughout the entire specification.

While this invention has been disclosed with reference to specificembodiments, it is apparent that other embodiments and variations ofthis invention may be devised by others skilled in the art withoutdeparting from the true spirit and scope of the invention.

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What is claimed is:
 1. A method for preventing or treating acute kidneyinjury, wherein said acute kidney injury is selected from the groupconsisting of myoglobinuric acute kidney injury, acute kidney injuryinduced by hypotensive shock, sepsis-associated acute kidney injury,toxic acute kidney injury, and acute kidney injury induced by iodinatedcontrast media, said method comprising administering to a subject inneed thereof a pharmaceutical composition comprising an effective amountof hepcidin or a biologically active fragment or homolog thereof,wherein said hepcidin or biologically active fragment or homolog thereofcomprises a sequence selected from the group consisting of SEQ ID NOs:1,2, 3, 4, and 5, thereby preventing or treating said acute kidney injury.2. The method of claim 1, wherein said acute kidney injury issepsis-associated acute kidney injury.
 3. The method of claim 1, whereinsaid method elicits an effect selected from the group consisting ofpreventing or treating oxidative stress associated injury in the kidney,inhibiting increased serum iron levels associated with acute kidneyinjury, inhibiting kidney iron accumulation, stimulating an increase insplenic non-heme iron levels, inhibiting a decrease in liver non-hemeiron, inhibiting increased hepcidin gene expression, inhibitingincreased endogenous serum hepcidin levels, inhibiting increased plasmacreatinine levels, inhibiting kidney tubular necrosis, inhibiting renalepithelial apoptosis, inhibiting oxidative stress, inhibitinginflammation, inhibiting infiltration of immune cells into the kidney,preventing or inhibiting an increase in acute kidney injury-inducedrenal ferroportin levels, inhibiting a decrease in H-ferritin levels,inhibiting infiltration of neutrophils and CD11b cells into the kidney,inhibiting splenic iron release, stimulating an increase in H-ferritinlevels, and inhibiting increases in interleukin-6 (IL-6) levels.
 4. Themethod of claim 1, wherein said hepcidin is administered in one or moredoses ranging from about 0.1 milligram/kilogram body weight (mg/kg bodywt.) to about 100 mg/kg body wt.
 5. The method of claim 4, wherein saidone or more doses is/are from about 1 mg/kg body wt. to about 10 mg/kgbody wt.
 6. The method of claim 5, wherein said one or more doses is/areselected from the group consisting of about 1.0, 1.5, 1.67, 2.0, 2.5,3.0, 3.33, 4.0, 4.5, 5.0, and 10 mg/kg body wt.
 7. The method of claim1, wherein said hepcidin is administered as a unit dose.
 8. The methodof claim 7, wherein said unit dose is selected from the group consistingof 50, 55, 60, 65, 70, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300,325, 350, 400, 450, 500, and 1,000 mg.
 9. The method of claim 1, whereinsaid subject is human.
 10. The method of claim 1, wherein saidcomposition is administered before said acute kidney injury.
 11. Themethod of claim 10, wherein said composition is administered at a rangeof about 1 minute to about 72 hours before said acute kidney injury. 12.The method of claim 10, wherein said composition is administered at atime selected from the group consisting of 1 minute, 10 minutes, 30minutes, 2 hours, 8 hours, 24 hours, 36 hours, 48 hours, and 72 hoursbefore said acute kidney injury.
 13. The method of claim 10, whereinsaid acute kidney injury is associated with a transplant in saidsubject.
 14. The method of claim 13, wherein said transplant is a kidneytransplant.
 15. The method of claim 10, wherein said acute kidney injuryis associated with cardiovascular surgery.
 16. The method of claim 1,further comprising administering at least one inducer of hepcidinsynthesis, levels, or activity to said subject.
 17. The method of claim16, wherein said inducer of hepcidin is selected from the groupconsisting of iron, iron-hepcidin complex, copper-hepcidin complex,metal-hepcidin complex, inhibitors of TMPRSS6, plant-derived flavonoids,cytokines, growth factors, steroid hormones, toll-like receptoractivation, tyrosine kinase inhibitors, mTOR inhibitors, bonemorphogenic protein 6 (BMP6), inducers of inflammation, erythroferrone,and minihepcidins.
 18. The method of claim 17, wherein said inhibitor ofTMPRSS6 is an antisense oligonucleotide or siRNA against Tmprss6, saidminihepcidin is PR73 or PR65, said cytokine is IL-6 or IL-22, saidgrowth factor is hepatocyte growth factor or epidermal growth factor,said steroid hormone is estrogen or testosterone, said tyrosine kinaseinhibitor is Sorafenib, and said mTOR inhibitor is rapamycin.
 19. Themethod of claim 1, wherein said pharmaceutical composition furthercomprises a pharmaceutically acceptable carrier, an additionaltherapeutic agent, or combinations thereof.